Abstract

The present invention relates to antibodies and related molecules that specifically bind to protective antigen of Bacillus anthracis (PA). Such antibodies have uses, for example, in the prevention and treatment of anthrax and anthrax toxin poisoning. The invention also relates to nucleic acid molecules encoding anti-PA antibodies, vectors and host cells containing these nucleic acids, and methods for producing the same.

This application refers to a “Sequence Listing” listed below, which is provided as a text file. The text file contains a document entitled “PF596P1C1.ST25.txt” (160,734 bytes, created Apr. 9, 2009), which is incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to antibodies and related molecules that specifically bind to the protective antigen (PA) of Bacillus anthracis. Such antibodies have uses, for example, in the prevention, detection and treatment of anthrax and/or anthrax related toxins. The invention also relates to nucleic acid molecules encoding anti-PA antibodies, vectors and host cells containing these nucleic acids, and methods for producing the same. The present invention relates to methods and compositions for preventing, detecting, diagnosing, treating or ameliorating anthrax and/or anthrax related toxins, comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that specifically bind to PA.

BACKGROUND OF THE INVENTION

Bacillus anthracis is a Gram-positive, aerobic, spore forming bacterium that is responsible for the deadly disease anthrax. There are three recognized routes of anthrax infection including cutaneous (through skin), gastrointestinal, and pulmonary (via inhalation) infection. Of the three ways to contract the disease, inhalation is the avenue that most frequently leads to the death of the patient.

Anthrax secretes a deadly three-component exotoxin which is comprised of three proteins, lethal factor (LF), edema factor (EF), and protective antigen (PA). The anthrax toxin is a bipartite toxin that contains A and B moieties, similar to that of diphtheria toxin and many clostridial toxins. The LF and EF proteins function as enzymatic A moieties of the toxin, while the PA protein functions as the B, or binding, moiety.

During the process of intoxication, PA binds to its cell surface receptor, (e.g., anthrax receptor (ATR) and/or capillary morphogenesis gene 2 (CMG2)) and is cleaved at the sequence RKKR (residues 193-196 of SEQ ID NO:2) by cell surface proteases such as furin. This cleavage releases a 20 kilodalton fragment of the PA protein, leaving a 63 kilodalton fragment of the PA protein bound to the cell surface (PA63). Some cleavage to the PA63 form may be mediated by serum proteases and occur prior to PA, in this case PA63, binding to the cell surface. Release of the 20 kilodalton PA fragment enables the PA63 fragment to multimerize into a heptameric ring structure and exposes a site on PA63 to which LF and EF bind with high affinity. The complex is then internalized by receptor-mediated endocytosis. Acidification of the vesicle causes conformational changes in the pA63 heptamer that result in transportation of LF and EF toxins across the endosomal membrane, after which they are released into the cytosol where they exert their cytotoxic effects. The edema factor (EF) component of edema toxin (EF+PA) is a calmodulin dependent adenylate cyclase whose action upsets cellular water homeostasis mechanisms, thereby resulting in swelling of infected tissues. The lethal factor (LF) moiety of lethal toxin (LF+PA) is a zinc metalloproteinase that inactivates mitogen activated protein kinase kinase in vitro. Lethal factor induces a hyperinflammatory condition in macrophages resulting in the production of proinflammatory cytokines including TNF-alpha and interleukin-1beta, which are responsible for shock and death of anthrax patients. For more detailed reviews of Bacillus Anthracis infection and anthrax toxin please see, e.g., Critical Reviews in Microbiology (2001) 27:167-200, Medical Progress (1999) 341:815-826, and Microbes and Infection (1999) 2:131-139, each of which are hereby incorporated by reference in their entireties.

There is a clear need, therefore, for identification and characterization of compositions, such as antibodies, that influence the biological activity of anthrax toxins.

The present invention relates to methods and compositions for preventing, treating or ameliorating anthrax disease and/or symptoms induced by anthrax related toxins (such as lethal toxin or edema toxin) comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that specifically bind to PA or a fragment or variant thereof. In specific embodiments, the present invention relates to methods and compositions for preventing, treating or ameliorating a disease or disorder associated with PA function, comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that specifically bind PA or a fragment or variant thereof.

In other embodiments, antibodies of the invention have a bactericidal effect on B. anthracis bacteria. By way of non-limiting example, antibodies of the invention may activate the classical complement pathway and/or enhance the activation of the alternative complement pathway which can lead to killing of bacterial cells. Alternatively, antibodies of the invention may opsonize B. anthracis bacteria. Opsonized bacteria then may be a target for antibody dependent cell-mediated cytotoxicty (ADCC). In another embodiment, antibodies of the invention may catalyze the generation of hydrogen peroxide from singlet molecular oxygen and water which chemical reaction results in the efficient killing of bacteria.

In specific embodiments, antibodies of the invention are administered in combination with other therapeutics or prophylactics such as a soluble form of an anthrax receptor (e.g., SEQ ID NO:3, described in Nature (2002) 414:225-229 (which is hereby incorporated by reference in its entirety), e.g., a polypeptide comprising amino acids 1-227 or 41-227 of SEQ ID NO:3) or a soluble form of the CMG2 receptor (SEQ ID NO:42, described in Scobie et al., Proceedings of the National Academy of Sciences USA (2003) 100:5170-5174 which is hereby incorporated by reference in its entirety, e.g., a polypeptide comprising amino acids 33-318 of SEQ ID NO:42). Other therapeutics or prophylactics that may be administered in combination with an antibody of the present invention include mutant forms of PA such as the EF/LF translocation deficient forms of PA described in International Publication Number WO01/82788 and in Science (2001) 292:695-697, both of which are hereby incorporated by reference in their entireties. Other therapeutics or prophylactics that may be administered in combination with an antibody of the present invention include peptide inhibitors that block LF binding to PA such as the P1 peptide, or its polyvalent form described in Nature Biotechnology (2002) 19:958-961 which is hereby incorporated by reference in its entirety. Still other therapeutics or prophylactics that may be administered in combination with an antibody of the present invention include, but are not limited to antibiotics, anthrax vaccines, antibodies immunoreactive with LF, EF or other protein moieties of Bacillus anthracis.

Another embodiment of the present invention includes the use of the antibodies of the invention as a diagnostic tool to monitor the presence of PA.

Single chain Fv's (scFvs) that specifically bind PA polypeptide (SEQ ID NOS:48-65) have been identified. Thus, the invention encompasses these scFvs, listed in Table 1. In addition, the invention encompasses cell lines engineered to express antibodies corresponding to these scFvs which are deposited with the American Type Culture Collection (“ATCC™”) as of the dates listed in Table 1 and given the ATCC™ Deposit Numbers identified in Table 1. The ATCC™ is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC™ deposit was made pursuant to the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Purposes of Patent Procedure.

Further, the present invention encompasses the polynucleotides encoding the scFvs, as well as the amino acid sequences encoding the scFvs. Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs (e.g., VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of the corresponding region of the recombinant antibody expressed by a cell line contained in an ATCC™ Deposit referred to in Table 1), that specifically bind to PA or fragments or variants thereof are also encompassed by the invention, as are nucleic acid molecules that encode these antibodies and/or molecules. In specific embodiments, the present invention encompasses antibodies, or fragments or variants thereof, that bind to an epitope that comprises the RKKR sequence of amino acid residues 193 to 196 of SEQ ID NO:2). In other embodiments, the antibodies of the invention bind an epitope of PA and occlude access of proteases to the RKKR cleavage site of PA (amino acid residues 193 to 196 of SEQ ID NO:2). In other embodiments, antibodies of the invention neutralize the ability of PA to bind to a cellular anthrax receptor, e.g., ATR (SEQ ID NO:3) or CMG2 (SEQ ID NO:42). In other embodiments, antibodies of the invention neutralize the ability of the PA (particularly the PA63 form of PA) to form oligomers, and more specifically to form heptamers. And in still other embodiments, antibodies of the invention neutralize the ability of PA (particularly the PA63 form of PA) to bind to either EF or LF (SEQ ID NOs:4 or 5, respectively).

The present invention also provides anti-PA antibodies that are coupled to a detectable label, such as an enzyme, a fluorescent label, a luminescent label, or a bioluminescent label. The present invention also provides anti-PA antibodies that are coupled to a therapeutic or cytotoxic agent. The present invention also provides anti-PA antibodies that which are coupled, directly or indirectly, to a radioactive material.

In further embodiments, the antibodies of the invention have a dissociation constant (KD) of 10−7 M or less. In preferred embodiments, the antibodies of the invention have a dissociation constant (KD) of 10−9 M or less.

In further embodiments, antibodies of the invention have an off rate (koff) of 10−3/sec or less. In preferred embodiments, antibodies of the invention have an off rate (koff) of 10−4/sec or less. In other preferred embodiments, antibodies of the invention have an off rate (koff) of 10−5/sec or less.

The present invention also provides for fusion proteins comprising an antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) of the invention, and a heterologous polypeptide (i.e., a polypeptide unrelated to an antibody or antibody domain). Nucleic acid molecules encoding these fusion proteins are also encompassed by the invention. A composition of the present invention may comprise, or alternatively consist of, one, two, three, four, five, ten, fifteen, twenty or more fusion proteins of the invention. Alternatively, a composition of the invention may comprise, or alternatively consist of, nucleic acid molecules encoding one, two, three, four, five, ten, fifteen, twenty or more fusion proteins of the invention.

The present invention also provides for a nucleic acid molecule(s), generally isolated, encoding an antibody (including molecules, such as scFvs, VH domains, or VL domains, that comprise, or alternatively consist of, an antibody fragment or variant thereof) of the invention. The present invention also provides a host cell transformed with a nucleic acid molecule of the invention and progeny thereof. The present invention also provides a method for the production of an antibody (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof) of the invention. The present invention further provides a method of expressing an antibody (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof) of the invention from a nucleic acid molecule. These and other aspects of the invention are described in further detail below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the ability of antibodies PWD0283 and PWD0587 to inhibit the binding of biotinylated PA to ATR.

FIG. 2 graphically depicts the binding of biotinylated-PA to CHO-K1 cells, J744.A murine macrophages, and human macrophages as determined by flow cytometry. The solid line depicts biotinylated —PA binding to cells; the dashed line depicts the background level.

FIG. 3 illustrates the ability of two antibodies PWD0283 and PWD0587 to inhibit pore formation by PA protein using the assay described in Example 5.

FIG. 5 illustrates the effect of prophylactic intravenous administration of PWD0283 and PWD0587 60 minutes prior to exposure of male Fisher 344 rats to Lethal Toxin. CAT002 is an isotype-matched (IgG1) negative control antibody. A single intravenous injection of PWD0283 or PWD0587 60 minutes prior to injection of lethal toxin provided 100% survival at 24 hours with no apparent ill effects. In contrast, a single injection of the negative control mAb, CAT002, provided no protection with 0% survival and an average TTM of 100 minutes. Vehicle or no study agent also provided no protection with 0% survival and an average TTM of 99 minutes and 91 minutes, respectively.

FIG. 6 shows the 14 day survival curves of the New Zealand White Rabbits (n=12) that received:

challenge via aerosol inhalation of approximately 195×LD50, of B. anthracis spores. Experimental details are described more fully in Example 11. Statistical p-values were obtained from a 2-sided log-rank test. The p-values for the comparison among all groups are <0.0001, regardless of inclusion or exclusion of the 40 mg/kg iv group in the analysis. The p-values marked in the graph are for the comparison versus the vehicle control group.

FIG. 7 shows the 28 day survival curves of cynomolgus monkeys (n=10 per group) that received no treatment (vehicle) or prophylactic treatment via subcutaneous administration of anti-PA monoclonal antibody PWD0587 (10, 20 or 40 mg/kg), two days prior to challenge via aerosol inhalation of approximately 186×LD50, of B. anthracis spores. Experimental details are described more fully in Example 12. Statistical p-values were obtained from a 2-sided log-rank test. The P values for the comparison among all groups are <0.0001. The P values marked in the graph are for the comparison versus the vehicle control group.

DETAILED DESCRIPTION OF THE INVENTIONDefinitions

The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody multimers and antibody fragments, as well as variants (including derivatives) of antibodies, antibody multimers and antibody fragments. Examples of molecules which are described by the term “antibody” herein include, but are not limited to: single chain Fvs (scFvs), Fab fragments, Fab′ fragments, F(ab′)2, disulfide linked Fvs (sdFvs), Fvs, and fragments comprising or alternatively consisting of, either a VL or a VH domain. The term “single chain Fv” or “scFv” as used herein refers to a polypeptide comprising a VL domain of antibody linked to a VH domain of an antibody.

Antibodies of the invention include, but are not limited to, monoclonal, multispecific, human or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), intracellularly-made antibodies (i.e., intrabodies), and epitope-binding fragments of any of the above. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. Preferably, an antibody of the invention comprises, or alternatively consists of, a VH domain, VH CDR, VL domain, or VL CDR having an amino acid sequence of any one of the cell lines in the ATCC™ Deposits referred, to referred to in Table 1, or a fragment or variant thereof. In a preferred embodiment, the immunoglobulin is an IgG1 isotype. In another preferred embodiment, the immunoglobulin is an IgG4 isotype. Immunoglobulins may have both a heavy and light chain. An array of IgG, IgE, IgM, IgD, IgA, and IgY heavy chains may be paired with a light chain of the kappa or lambda forms. Antibodies of the invention may also include multimeric forms of antibodies. For example, antibodies of the invention may take the form of antibody dimers, trimers, or higher-order multimers of monomeric immunoglobulin molecules. Dimers of whole immunoglobulin molecules or of F(ab′)2 fragments are tetravalent, whereas dimers of Fab fragments or scFv molecules are bivalent. Individual monomers withon an antibody multimer may be identical or different, i.e., they may be heteromeric or homomeric antibody multimers. For example, individual antibodies within a multimer may have the same or different binding specificities.

Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers, and other higher-order antibody multimers. Alternatively, antibody homodimers may be formed through chemical linkage techniques known in the art. For example, heterobifunctional crosslinking agents including, but not limited to, SMCC [succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate] and SATA [N-succinimidyl S-acethylthio-acetate] (available, for example, from Pierce Biotechnology, Inc. (Rockford, Ill.)) can be used to form antibody multimers. An exemplary protocol for the formation of antibody homodimers is given in Ghetie et al., Proceedings of the National Academy of Sciences USA (1997) 94:7509-7514, which is hereby incorporated by reference in its entirety. Antibody homodimers can be converted to Fab′2 homodimers through digestion with pepsin. Another way to form antibody homodimers is through the use of the autophilic T15 peptide described in Zhao and Kohler, The Journal of Immunology (2002) 25:396-404, which is hereby incorporated by reference in its entirety.

Alternatively, antibodies can be made to multimerize through recombinant DNA techniques. IgM and IgA naturally form antibody multimers through the interaction with the mature J chain polypeptide (e.g., SEQ ID NO:44). Non-IgA or non-IgM molecules, such as IgG molecules, can be engineered to contain the J chain interaction domain of IgA or IgM, thereby conferring the ability to form higher order multimers on the non-IgA or non-IgM molecules. (see, for example, Chintalacharuvu et al., (2001) Clinical Immunology 101:21-31. and Frigerio et al., (2000) Plant Physiology 123:1483-94., both of which are hereby incorporated by reference in their entireties.) IgA dimers are naturally secreted into the lumen of mucosa-lined organs. This secretion is mediated through interaction of the J chain with the polymeric IgA receptor (pIgR) on epithelial cells. If secretion of an IgA form of an antibody (or of an antibody engineered to contain a J chain interaction domain) is not desired, it can be greatly reduced by expressing the antibody molecule in association with a mutant J chain that does not interact well with pIgR (e.g., SEQ ID NOS:45-47; Johansen et al., The Journal of Immunology (2001) 167:5185-5192 which is hereby incorporated by reference in its entirety). Expression of an antibody with one of these mutant J chains will reduce its ability to bind to the polymeric IgA receptor on epithelial cells, thereby reducing transport of the antibody across the epithelial cell and its resultant secretion into the lumen of mucosa lined organs. ScFv dimers can also be formed through recombinant techniques known in the art; an example of the construction of scFv dimers is given in Goel et al., (2000) Cancer Research 60:6964-6971 which is hereby incorporated by reference in its entirety. Antibody multimers may be purified using any suitable method known in the art, including, but not limited to, size exclusion chromatography.

By “isolated antibody” is intended an antibody removed from its native environment. Thus, an antibody produced by, purified from and/or contained within a hybridoma and/or a recombinant host cell is considered isolated for purposes of the present invention.

Unless otherwise defined in the specification, specific binding by an antibody to PA means that an antibody binds PA but does not significantly bind to (i.e., cross react with) proteins other than PA, such as other proteins in the same family of proteins). An antibody that binds PA protein and does not cross-react with other proteins is not necessarily an antibody that does not bind said other proteins in all conditions; rather, the PA-specific antibody of the invention preferentially binds PA compared to its ability to bind said other proteins such that it will be suitable for use in at least one type of assay or treatment, i.e., give low background levels or result in no unreasonable adverse effects in treatment. It is well known that the portion of a protein bound by an antibody is known as the epitope. An epitope may either be linear (i.e., comprised of sequential amino acids residues in a protein sequences) or conformational (i.e., comprised of one or more amino acid residues that are not contiguous in the primary structure of the protein but that are brought together by the secondary, tertiary or quaternary structure of a protein). Given that PA-specific antibodies bind to epitopes of PA, an antibody that specifically binds PA may or may not bind fragments of PA and/or variants of PA (e.g., proteins that are at least 90% identical to PA) depending on the presence or absence of the epitope bound by a given PA-specific antibody in the PA fragment or variant. Likewise, PA-specific antibodies of the invention may bind species orthologues of PA (including fragments thereof) depending on the presence or absence of the epitope recognized by the antibody in the orthologue. Additionally, PA-specific antibodies of the invention may bind modified forms of PA, for example, PA fusion proteins. In such a case when antibodies of the invention bind PA fusion proteins, the antibody must make binding contact with the PA moiety of the fusion protein in order for the binding to be specific. Antibodies that specifically bind to PA can be identified, for example, by immunoassays or other techniques known to those of skill in the art, e.g., the immunoassays described in the Examples below.

Antibodies of the invention may also include multimeric forms of antibodies. For example, antibodies of the invention may take the form of antibody dimers, trimers, or higher-order multimers of monomeric immunoglobulin molecules. Dimers of whole immunoglobulin molecules or of F(ab′)2 fragments are tetravalent, whereas dimers of Fab fragments or scFv molecules are bivalent. Individual monomers within an antibody multimer may be identical or different, i.e., they may be heteromeric or homomeric antibody multimers. For example, individual antibodies within a multimer may have the same or different binding specificities. Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers, and other higher-order antibody multimers. Alternatively, antibody homodimers may be formed through chemical linkage techniques known in the art. For example, heterobifunctional crosslinking agents including, but not limited to, SMCC [succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate] and SATA [N-succinimidyl S-acethylthio-acetate] (available, for example, from Pierce Biotechnology, Inc. (Rockford, Ill.)) can be used to form antibody multimers. An exemplary protocol for the formation of antibody homodimers is given in Ghetie et al., Proceedings of the National Academy of Sciences USA (1997) 94:7509-7514, which is hereby incorporated by reference in its entirety. Antibody homodimers can be converted to Fab′2 homodimers through digestion with pepsin. Alternatively, antibodies can be made to multimerize through recombinant DNA techniques. IgM and IgA naturally form antibody multimers through the interaction with the J chain polypeptide. Non-IgA or non-IgM molecules, such as IgG molecules, can be engineered to contain the J chain interaction domain of IgA or IgM, thereby conferring the ability to form higher order multimers on the non-IgA or non-IgM molecules. (see, for example, Chintalacharuvu et al., (2001) Clinical Immunology 101:21-31 and Frigerio et al., (2000) Plant Physiology 123:1483-94, both of which are hereby incorporated by reference in their entireties.) ScFv dimers can also be formed through recombinant techniques known in the art; an example of the construction of scFv dimers is given in Goel et al., (2000) Cancer Research 60:6964-6971, which is hereby incorporated by reference in its entirety. Antibody multimers may be purified using any suitable method known in the art, including, but not limited to, size exclusion chromatography.

The term “variant” as used herein refers to a polypeptide that possesses a similar or identical amino acid sequence as a PA polypeptide, a fragment of a PA polypeptide, an anti-PA antibody or antibody fragment thereof. A variant having a similar amino acid sequence refers to a polypeptide that satisfies at least one of the following: (a) a polypeptide comprising, or alternatively consisting of, an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence of PA polypeptide (SEQ ID NO:2), a fragment of a PA polypeptide, an anti-PA antibody or antibody fragment thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence of any one or more scFvs or recombinant antibodies expressed by the cell lines in the ATCC™ Deposits referred to in Table 1) described herein; (b) a polypeptide encoded by a nucleotide sequence, the complementary sequence of which hybridizes under stringent conditions to a nucleotide sequence encoding PA (SEQ ID NO:2), a fragment of a PA polypeptide, an anti-PA antibody or antibody fragment thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence of any one of the scFvs referred to in Table 1), described herein, of at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 30 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, or at least 150 amino acid residues; and (c) a polypeptide encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99%, identical to the nucleotide sequence encoding a PA polypeptide, a fragment of a PA polypeptide, an anti-PA antibody or antibody fragment thereof (including a VH domain, VHCDR, VL domain, or VLCDR having an amino acid sequence of any one or more scFvs or recombinant antibodies expressed by the cell lines in the ATCC™ Deposits referred to in Table 1), described herein. A polypeptide with similar structure to a PA polypeptide, a fragment of a PA polypeptide, an anti-PA antibody or antibody fragment thereof, described herein refers to a polypeptide that has a similar secondary, tertiary or quaternary structure of a PA polypeptide, a fragment of a PA polypeptide, an anti-PA antibody, or antibody fragment thereof, described herein. The structure of a polypeptide can determined by methods known to those skilled in the art, including but not limited to, X-ray crystallography, nuclear magnetic resonance, and crystallographic electron microscopy. Preferably, a variant PA polypeptide, a variant fragment of a PA polypeptide, or a variant anti-PA antibody and/or antibody fragment possesses similar or identical function and/or structure as the reference PA polypeptide, the reference fragment of a PA polypeptide, or the reference anti-PA antibody and/or antibody fragment, respectively.

To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions/total number of positions ×100%). In one embodiment, the two sequences are the same length.

The determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993). The BLASTn and BLASTx programs of Altschul, et al. J. Mol. Biol. 215:403-410 (1990) have incorporated such an algorithm. BLAST nucleotide searches can be performed with the BLASTn program (score=100, wordlength=12) to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTx program (score=50, wordlength=3) to obtain amino acid sequences homologous to a protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. Nucleic Acids Res. 25:3589-3402 (1997). Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used.

Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0) which is part of the GCG sequence alignment software package has incorporated such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti Comput. Appl. Biosci., 10:3-5 (1994); and FASTA described in Pearson and Lipman Proc. Natl. Acad. Sci. 85:2444-8 (1988). Within FASTA, ktup is a control option that sets the sensitivity and speed of the search.

The term “derivative” as used herein, refers to a variant polypeptide of the invention that comprises, or alternatively consists of, an amino acid sequence of a PA polypeptide, a fragment of a PA polypeptide, or an antibody of the invention that specifically binds to a PA polypeptide, which has been altered by the introduction of amino acid residue substitutions, deletions or additions. The term “derivative” as used herein also refers to a PA polypeptide, a fragment of a PA polypeptide, an antibody that specifically binds to a PA polypeptide which has been modified, e.g., by the covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, a PA polypeptide, a fragment of a PA polypeptide, or an anti-PA antibody, may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. A derivative of a PA polypeptide, a fragment of a PA polypeptide, or an anti-PA antibody, may be modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Further, a derivative of a PA polypeptide, a fragment of a PA polypeptide, or an anti-PA antibody, may contain one or more non-classical amino acids. A polypeptide derivative possesses a similar or identical function as a PA polypeptide, a fragment of a PA polypeptide, or an anti-PA antibody, described herein.

The term “host cell” as used herein refers to the particular subject cell transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

Antibodies of the present invention are preferably provided in an isolated form, and preferably are substantially purified. By “isolated” is intended an antibody removed from its native environment. Thus, for example, an antibody produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention.

Antibody Structure

The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kilodalton) and one “heavy” chain (about 50-70 kilodalton). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. Herein the terms “heavy chain” and “light chain” refer to the heavy and light chains of an antibody unless otherwise specified. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light/heavy chain pair form the antibody binding site.

Thus, an intact IgG antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are the same.

The chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs. The CDRs from the heavy and the light chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989).

Production of bispecific antibodies can be a relatively labor intensive process compared with production of conventional antibodies and yields and degree of purity are generally lower for bispecific antibodies. Bispecific antibodies do not exist in the form of fragments having a single binding site (e.g., Fab, Fab′, and Fv).

Anti-PA Antibodies

Using phage display technology, single chain antibody molecules (“scFvs”) that specifically bind to PA (or fragments or variants thereof) have been identified (Example 1). Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs (e.g., VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of the corresponding region of the antibody expressed by a cell line contained in an ATCC™ Deposit referred to in Table 1), that specifically bind to PA (or fragments or variants thereof) are also encompassed by the invention, as are nucleic acid molecules that encode these scFvs, and/or molecules.

In particular, the invention relates to scFvs comprising, or alternatively consisting of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 48-56, preferably SEQ ID NOs:50 and 53 as referred to in Table 1 below. Molecules comprising, or alternatively consisting of, fragments or variants of these scFvs (e.g., VH domains, VH CDRs, VL domains, or VL CDRs having an amino acid sequence of any one of those referred to in Table 1), that specifically bind to PA are also encompassed by the invention, as are nucleic acid molecules that encode these scFvs, and/or molecules (e.g., SEQ ID NOs:57-65).

The present invention provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that specifically bind to a polypeptide or a polypeptide fragment of PA. In particular, the invention provides antibodies corresponding to the scFvs referred to in Table 1. Such scFvs may routinely be “converted” to immunoglobulin molecules by inserting, for example, the nucleotide sequences encoding the VH and/or VL domains of the scFv into an expression vector containing the constant domain sequences and engineered to direct the expression of the immunoglobulin molecule, as described in more detail in Example 6 below.

NS0 cell lines that express IgG1 antibodies that comprise the VH and VL domains of scFvs of the invention have been deposited with the American Type Culture Collection (“ATCC™”) on the dates listed in Table 1 and given the ATCC™ Deposit Numbers identified in Table 1. The ATCC™ is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC™ deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure.

Accordingly, in one embodiment, the invention provides antibodies that comprise the VH and VL domains of scFvs of the invention.

In a preferred embodiment, an antibody of the invention is the antibody expressed by cell line NSO PA 2973 (PWD0587) #240-22 (See Table 1).

Antibodies of the present invention bind PA polypeptide or fragments or variants thereof. The following section describes the PA polypeptides, fragments and variants that may be bound by the antibodies of the invention in more detail.

The PA protein is a 764 amino acid protein (SEQ ID NO:2) comprising a signal sequence from amino acid residues 1-29, and a 735 amino acid secreted protein which undergoes further process upon binding to an anthrax receptor, (e.g., ATR or CMG2) on the cell surface. The 735 amino acid secreted protein, also known as PA83 because it has a molecular weight of approximately 83 kilodaltons, has a structure that is largely made up of antiparallel beta pleated sheets with only a few short alpha-helices. The protein can be divided into four domains: Domain I (amino acid residues 30-287 of SEQ ID NO:2), Domain II (amino acid residues 288-516 of SEQ ID NO:2), Domain III (amino acid residues 517-624 of SEQ ID NO:2), and Domain IV (amino acid residues 625-764) of SEQ ID NO:2). In its native form, Domain I contains two calcium ions and the protease cleavage site RKKR at amino acid residues 193-196 of SEQ ID NO:2. Thus, Domain I contains the entire 20 kilodalton fragment (PA20, amino acid residues 30-196 of SEQ ID NO:2) that is cleaved off of PA upon binding to an anthrax receptor (e.g., ATR or CMG2) at the cell surface. That portion of Domain I that remains after cleavage of PA20 forms the N terminus of active PA63 and may be involved in binding LF and EF. Domain II is the heptamerization domain and also contains a large flexible loop that is implicated in membrane insertion. Domain III, is small and its function is not clearly understood. Domain IV is the receptor binding domain.

Thus, in specific embodiments, antibodies of the invention may bind the intact 735 amino acid secreted form of PA (PA83), polypeptides that comprise or alternatively consist of the PA63 protein, the PA20 fragment, and/or any one or more of domains I, II, III, or IV. In preferred embodiments, antibodies of the invention bind PA83 and prevent its cleavage of the PA20 fragment from the PA63 fragment by proteases. In other embodiments, antibodies of the invention bind the PA63 form of PA and prevent oligomerization, and in particular heptamerization of PA63.

In certain embodiments, the antibodies of the present invention specifically bind PA polypeptide. An antibody that specifically binds PA may, in some embodiments, bind fragments, variants (including species orthologs of PA), multimers or modified forms of PA. For example, an antibody specific for PA may bind the PA moiety of a fusion protein comprising all or a portion of PA.

PA proteins may be found as monomers or multimers (i.e., dimers, trimers, tetramers, and higher multimers). Accordingly, the present invention relates to antibodies that bind PA proteins found as monomers or as part of multimers. In specific embodiments, antibodies of the invention bind PA monomers, dimers, trimers or heptamers. In additional embodiments, antibodies of the invention bind at least dimers, at least trimers, or at least tetramers containing one or more PA polypeptides.

Antibodies of the invention may bind PA homomers or heteromers. As used herein, the term homomer, refers to a multimer containing only PA proteins of the invention (including PA fragments such as PA63, variants, and fusion proteins, as described herein). These homomers may contain PA proteins having identical or different polypeptide sequences. In a specific embodiment, a homomer of the invention is a multimer containing only PA proteins having an identical polypeptide sequence. In another specific embodiment, antibodies of the invention bind PA homomers containing PA proteins having different polypeptide sequences. In specific embodiments, antibodies of the invention bind a PA homodimer (e.g., containing PA proteins having identical or different polypeptide sequences). In additional embodiments, antibodies of the invention bind at least a homodimer, at least a homotrimer, or at least a homotetramer of PA.

As used herein, the term heteromer refers to a multimer containing heterologous proteins (i.e., proteins containing polypeptide sequences that do not correspond to a polypeptide sequences encoded by the PA gene) in addition to the PA proteins of the invention. In a specific embodiment, antibodies of the invention bind a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the antibodies of the invention bind at least a heterodimer, at least a heterotrimer, or at least a-heterotetramer containing one or more PA polypeptides.

In specific embodiments, antibodies of the present invention bind a PA heteroheptamer.

Antibodies of the invention may bind PA multimers that are the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation. Thus, in one embodiment, antibodies of the invention may bind PA multimers, such as, for example, homoheptamers, that are formed when PA proteins (such as PA63 polypeptide monomers) contact one another in solution. In another embodiment, antibodies of the invention may bind heteromultimers, such as, for example, heteroheptamers, that are formed when proteins of the invention contact antibodies to the PA polypeptides (including antibodies to the heterologous polypeptide sequence in a fusion protein) in solution. In other embodiments, multimers bound by one or more antibodies of the invention are formed by covalent associations with and/or between the PA proteins of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence of the protein (e.g., the polypeptide sequence recited in SEQ ID NO:2). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences of the proteins which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a PA fusion protein. In one example, covalent associations are between the heterologous sequence contained in a fusion protein (see, e.g., U.S. Pat. No. 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in a PA-Fc or PA-human serum albumin (PA-HSA) fusion protein (as described herein).

Antibodies of the invention may bind PA multimers generated using chemical techniques known in the art. For example, proteins desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, multimers that antibodies of the invention may bind can be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the polypeptide sequence of the proteins desired to be contained in the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Further, proteins that antibodies of the invention may bind can be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide sequence of the protein and techniques known in the art may be applied to generate multimers containing one or more of these modified proteins (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the protein components desired to be contained in the multimer that antibodies of the invention may bind (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).

Alternatively, multimers that antibodies of the invention may bind can be generated using genetic engineering techniques known in the art. In one embodiment, proteins contained in multimers that may be bound by one or more antibodies of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In a specific embodiment, polynucleotides coding for a homodimer that may be bound by one or more antibodies of the invention are generated by ligating a polynucleotide sequence encoding a PA polypeptide to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant PA polypeptides which contain a transmembrane domain and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, two or more PA polypeptides are joined through synthetic linkers (e.g., peptide, carbohydrate or soluble polymer linkers). Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple PA polypeptides separated by peptide linkers may be produced using conventional recombinant DNA technology. In specific embodiments, antibodies of the invention bind proteins comprising multiple PA polypeptides separated by peptide linkers.

Another method for preparing multimer PA polypeptides involves use of PA polypeptides fused to a leucine zipper or isoleucine polypeptide sequence. Leucine zipper domains and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric PA proteins are those described in PCT application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins comprising a soluble PA polypeptide fused to a peptide that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric PA is recovered from the culture supernatant using techniques known in the art. In specific embodiments, antibodies of the invention bind PA-leucine zipper fusion protein monomers and/or PA-leucine zipper fusion protein multimers.

Antibodies that bind PA receptor polypeptides may bind them as isolated polypeptides or in their naturally occurring state. By “isolated polypeptide” is intended a polypeptide removed from its native environment. Thus, a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention. Also intended as an “isolated polypeptide” are polypeptides that have been purified, partially or substantially, from a recombinant host cell. For example, a recombinantly produced version of the PA polypeptide may be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). Thus, antibodies of the present invention may bind recombinantly and/or naturally produced PA polypeptides. In a specific embodiment, antibodies of the present invention bind a PA secreted by a cell, preferably a bacterial cell, comprising a polynucleotide encoding amino acids 1 to 764 of SEQ ID NO:2 operably associated with a regulatory sequence that controls gene expression. In a specific embodiment, antibodies of the present invention bind PA purified from a bacterial cell culture, wherein said PA is encoded by a polynucleotide encoding amino acids 1 to 764 of SEQ ID NO:2 operably associated with a regulatory sequence that controls expression of said polynucleotide. In other specific embodiments, antibodies of the present invention bind a PA polypeptide expressed by a cell comprising a polynucleotide encoding amino acids 197 to 764 of SEQ ID NO:2 operably associated with a regulatory sequence that controls gene expression. In still other embodiments, antibodies of the present invention bind a PA polypeptide expressed by a cell comprising a polynucleotide encoding amino acids 625 to 764 of SEQ ID NO:2 operably associated with a regulatory sequence that controls gene expression.

Antibodies of the present invention that may bind PA polypeptide fragments comprising or alternatively, consisting of, an amino acid sequence contained in SEQ ID NO:2. Protein fragments may be “free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Antibodies of the present invention may bind polypeptide fragments, including, for example, fragments that comprise or alternatively, consist of from about amino acid residues: 1 to 29, 30 to 59, 60 to 89, 90 to 119, 120 to 149, 150 to 175, 176 to 196, 197 to 226, 227 to 256, 257 to 287, 288 to 312, 313 to 337, 338 to 362, 363 to 387, 388 to 412, 413 to 437, 438 to 462, 463 to 487, 488 to 516, 517 to 542, 543 to 569, 570 to 569, 570 to 596, 597 to 624, 625 to 652, 653 to 680, 681 to 708, 709 to 736, and/or 737 to 764 of SEQ ID NO:2. In this context “about” includes the particularly recited value, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Moreover, polypeptide fragments that antibodies of the invention may bind can be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175 or 200 amino acids in length. In this context “about” includes the particularly recited value, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.

Preferably, antibodies of the present invention bind polypeptide fragments selected from the group: a polypeptide comprising or alternatively, consisting of, the full length PA polypeptide (amino acid residues 1 to 764 in SEQ ID NO:2); a polypeptide comprising or alternatively, consisting of, the secreted form of PA (amino acid residues 30 to 764 in SEQ ID NO:2); a polypeptide comprising or alternatively, consisting of, the PA20 fragment (amino acid residues from about 30 to about 196 in SEQ ID NO:2); a polypeptide comprising or alternatively, consisting of, the PA63 fragment (amino acid residues from about 197 to about 764 in SEQ ID NO:2); a polypeptide comprising or alternatively, consisting of, PA domain I (amino acid residues 30 to 287 of SEQ ID NO:2); a polypeptide comprising or alternatively, consisting of, PA domain II (amino acid residues 288 to 516 of SEQ ID NO:2); a polypeptide comprising or alternatively, consisting of, PA domain 111 (amino acid residues 517 to 624 of SEQ ID NO:2); a polypeptide comprising or alternatively, consisting of, PA domain IV (amino acid residues 625 to 764 of SEQ ID NO:2); a polypeptide comprising or alternatively, consisting of, fragment of the predicted mature PA polypeptide; and a polypeptide comprising, or alternatively, consisting of, one, two, three, four or more, epitope bearing portions of the PA receptor protein. In additional embodiments, the polypeptide fragments of the invention comprise, or alternatively, consist of, any combination of 1, 2, 3, 4, 5, 6, 7, or all 8 of the above members. The amino acid residues constituting these domains may vary slightly (e.g., by about 1 to about 15 amino acid residues) depending on the criteria used to define each domain.

Domain I contains the proteolytic cleavage site. When the secreted form of PA is cleaved at this site, a 20 kilodalton fragment (PA20) is released from PA, generating the biologically active 63 kilodalton PA63 fragment. Thus, in specific embodiments antibodies of the invention bind an epitope at or near this cleavage site and prevent the cleavage of the secreted form of PA that results in the generation of PA20 and PA63. In specific embodiments, antibodies of the invention that prevent cleavage of PA into PA20 and PA63 may bind one or more PA peptides (as well as the native amino acid secreted form of the protein, PA83, see, e.g., Example 2) selected from the group consisting of: (a) amino acid residues 190 to 209 of SEQ ID NO:2; (b) amino acid residues 181 to 201 of SEQ ID NO:2; (c) amino acid residues 198 to 212 of SEQ ID NO:2; (d) amino acid residues 196 to 212 of SEQ ID NO:2; (e) amino acid residues 194 to 212 of SEQ ID NO:2; (f) amino acid residues 192 to 212 of SEQ ID NO:2; (g) amino acid residues 190 to 212 of SEQ ID NO:2; (h) amino acid residues 188 to 212 of SEQ ID NO:2; (i) amino acid residues 186 to 212 of SEQ ID NO:2; (j) amino acid residues 184 to 212 of SEQ ID NO:2; and (k) amino acid residues 181 to 195 of SEQ ID NO:2.

Domain IV of PA is important for interactions between PA and its receptor (e.g., ATR (SEQ ID NO:3) or CMG2 (SEQ ID NO:42)). Accordingly, in preferred embodiments, antibodies of the present invention bind PA polypeptide fragments comprising, or alternatively consisting of amino acid residues 625 to 764 of SEQ ID NO:2. In preferred embodiments, the antibodies of the invention that bind all or a portion of domain IV of PA prevent PA from binding to ATR and/or CMG2. In other preferred embodiments, the antibodies of the invention that bind all or a portion of domain IV of PA protect cells from death induced by anthrax toxins.

Antibodies of the invention may also bind fragments comprising, or alternatively, consisting of structural or functional attributes of PA. Such fragments include amino acid residues that comprise alpha-helix and alpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheet-forming regions (“beta-regions”), turn and turn-forming regions (“turn-regions”), coil and coil-forming regions (“coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, surface forming regions, and high antigenic index regions (i.e., containing four or more contiguous amino acids having an antigenic index of greater than or equal to 1.5, as identified using the default parameters of the Jameson-Wolf program) of complete (i.e., full-length) PA. Certain preferred regions are those set out in Table 2 and include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence depicted in (SEQ ID NO:2), such preferred regions include; Garnier-Robson predicted alpha-regions, beta-regions, turn-regions, and coil-regions; Chou-Fasman predicted alpha-regions, beta-regions, and turn-regions; Kyte-Doolittle predicted hydrophilic regions; Eisenberg alpha and beta amphipathic regions; Emini surface-forming regions; and Jameson-Wolf high antigenic index regions, as predicted using the default parameters of these computer programs.

The data representing the structural or functional attributes of PA set forth in Table 2, as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. Column I represents the results of a Garnier-Robson analysis of alpha helical regions; Column II represents the results of a Chou-Fasman analysis of alpha helical regions; Column 111 represents the results of a Garnier Robson analysis of beta sheet regions; Column IV represents the results of a Chou-Fasman analysis of beta sheet regions; Column V represents the results of a Garnier Robson analysis of turn regions; Column VI represents the results of a Chou-Fasman analysis of turn regions; Column VII represents the results of a Garnier Robson analysis of coil regions; Column VIII represents a Kyte-Doolittle hydrophilicity plot; Column; Column IX represents the results of an Eisenberg analysis of alpha amphipathic regions; Column X represents the results of an Eisenberg analysis of beta amphipathic regions; Column XI represents the results of a Karplus-Schultz analysis of flexible regions; Column XII represents the Jameson-Wolf antigenic index score; and Column XIII represents the Emini surface probability plot.

In a preferred embodiment, the data presented in columns VIII, XII, and XIII of Table 2 can be used to determine regions of PA which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, XII, and/or XIII by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.

The above-mentioned preferred regions set out in Table 2 include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in SEQ ID NO:2. As set out in Table 2, such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming regions. Among preferred polypeptide fragments bound by one or more antibodies of the invention are those that comprise regions of PA that combine several structural features, such as several (e.g., 1, 2, 3, or 4) of the same or different region features set out above and in Table 2.

TABLE 2

Res

Position

I

II

III

IV

V

VI

VII

VIII

IX

X

XI

XII

XIII

Met

1

A

A

.

.

.

.

.

1.59

.

.

.

0.75

2.90

Lys

2

A

A

.

.

.

.

.

1.12

.

.

.

0.75

4.54

Lys

3

A

A

.

B

.

.

.

0.70

.

.

.

0.75

2.64

Arg

4

A

A

.

B

.

.

.

0.20

.

.

.

0.75

2.20

Lys

5

A

A

.

B

.

.

.

0.38

.

.

F

0.75

0.77

Val

6

.

A

B

B

.

.

.

0.17

.

.

.

0.60

0.60

Leu

7

.

A

B

B

.

.

.

−0.48

.

.

.

−0.30

0.25

Ile

8

.

A

B

B

.

.

.

−1.11

.

.

.

−0.60

0.12

Pro

9

.

A

B

B

.

.

.

−2.03

*

.

.

−0.60

0.17

Leu

10

.

A

B

B

.

.

.

−2.38

.

*

.

−0.60

0.17

Met

11

A

A

.

B

.

.

.

−1.83

.

.

.

−0.60

0.32

Ala

12

A

A

.

B

.

.

.

−1.91

.

.

.

−0.60

0.30

Leu

13

A

A

.

B

.

.

.

−1.83

.

.

.

−0.60

0.26

Ser

14

.

A

B

B

.

.

.

−2.48

.

.

.

−0.60

0.21

Thr

15

.

.

B

B

.

.

.

−1.97

.

.

.

−0.60

0.16

Ile

16

.

.

B

B

.

.

.

−1.67

.

.

.

−0.60

0.25

Leu

17

.

.

B

B

.

.

.

−1.39

.

.

.

−0.60

0.25

Val

18

.

.

B

B

.

.

.

−0.92

.

.

.

−0.60

0.25

Ser

19

.

.

B

B

.

.

.

−0.62

.

*

F

−0.36

0.36

Ser

20

.

.

.

.

.

T

C

−1.12

.

*

F

0.33

0.70

Thr

21

.

.

.

.

.

T

C

−0.23

.

*

F

0.42

0.78

Gly

22

.

.

.

.

.

T

C

−0.28

.

*

F

1.56

1.01

Asn

23

.

.

.

.

.

T

C

−0.31

.

*

F

0.90

0.56

Leu

24

A

A

.

.

.

.

.

−0.01

.

.

.

0.06

0.27

Glu

25

A

A

.

.

.

.

.

−0.30

.

.

.

−0.03

0.47

Val

26

A

A

.

.

.

.

.

0.01

.

*

.

−0.12

0.30

Ile

27

A

A

.

.

.

.

.

−0.50

.

*

.

0.39

0.63

Gln

28

A

A

.

.

.

.

.

−0.46

*

*

.

0.30

0.27

Ala

29

A

A

.

.

.

.

.

0.36

*

*

.

0.30

0.72

Glu

30

A

A

.

.

.

.

.

0.36

*

*

.

0.45

1.79

Val

31

A

A

.

.

.

.

.

1.21

*

*

F

0.90

1.79

Lys

32

A

A

.

.

.

.

.

2.21

*

*

F

0.90

2.84

Gln

33

A

A

.

.

.

.

.

1.40

*

*

F

0.90

3.21

Glu

34

A

A

.

.

.

.

.

1.18

*

*

F

0.90

3.57

Asn

35

A

A

.

.

.

.

.

1.18

*

*

F

0.90

1.47

Arg

36

A

A

.

.

.

.

.

2.03

*

*

F

0.60

1.37

Leu

37

A

A

.

.

.

.

.

1.69

*

.

F

0.90

1.37

Leu

38

A

A

.

.

.

.

.

1.69

*

.

F

0.94

1.14

Asn

39

.

.

.

.

.

T

C

1.39

*

.

F

2.18

1.01

Glu

40

A

.

.

.

.

T

.

1.09

*

.

F

2.02

1.64

Ser

41

.

.

.

.

.

T

C

0.68

*

.

F

2.86

2.66

Glu

42

.

.

.

.

T

T

.

1.49

.

.

F

3.40

2.22

Ser

43

.

.

.

.

T

T

.

1.96

.

.

F

3.06

2.22

Ser

44

.

.

.

.

T

T

.

1.14

.

.

F

2.72

1.64

Ser

45

.

.

.

.

T

T

.

0.33

.

.

F

1.93

0.78

Gln

46

.

.

B

.

.

T

.

0.29

.

.

F

0.59

0.48

Gly

47

.

.

B

B

.

.

.

0.04

.

.

F

−0.45

0.35

Leu

48

.

.

B

B

.

.

.

0.10

.

.

F

−0.45

0.41

Leu

49

.

.

B

B

.

.

.

−0.30

.

.

.

−0.60

0.37

Gly

50

.

.

B

B

.

.

.

−0.30

.

.

.

−0.60

0.33

Tyr

51

.

.

B

B

.

.

.

−0.30

.

.

.

−0.60

0.53

Tyr

52

.

.

B

B

.

.

.

−0.77

.

.

.

−0.45

1.08

Phe

53

.

.

B

B

.

.

.

0.04

.

.

.

−0.60

0.90

Ser

54

.

.

B

.

.

.

.

0.16

.

*

.

−0.40

0.92

Asp

55

.

A

B

.

.

.

.

0.50

.

*

.

−0.60

0.51

Leu

56

.

A

B

.

.

.

.

0.16

.

*

.

−0.45

1.02

Asn

57

.

A

.

.

T

.

.

0.19

.

*

.

0.10

0.77

Phe

58

.

A

.

.

T

.

.

0.29

.

*

.

0.10

0.71

Gln

59

A

A

.

.

.

.

.

−0.27

.

*

.

−0.60

0.85

Ala

60

.

A

B

B

.

.

.

−1.12

.

*

.

−0.60

0.39

Pro

61

.

A

B

B

.

.

.

−0.62

.

*

.

−0.60

0.34

Met

62

.

A

B

B

.

.

.

−0.92

.

*

.

−0.60

0.28

Val

63

.

A

B

B

.

.

.

−0.52

.

.

.

−0.60

0.37

Val

64

.

.

B

B

.

.

.

−0.83

.

.

.

−0.60

0.32

Thr

65

.

.

B

B

.

.

.

−0.56

.

.

F

−0.20

0.47

Ser

66

.

.

B

B

.

.

.

−0.69

.

.

F

0.05

0.92

Ser

67

.

.

B

B

.

.

.

−0.09

.

.

F

0.75

1.22

Thr

68

.

.

B

.

.

T

.

−0.04

.

.

F

2.00

1.42

Thr

69

.

.

.

.

T

T

.

0.51

.

*

F

2.50

0.87

Gly

70

.

.

.

.

T

T

.

−0.07

.

*

F

2.25

0.87

Asp

71

.

.

B

.

.

T

.

0.02

.

*

F

1.00

0.42

Leu

72

.

.

B

.

.

.

.

0.02

.

*

F

0.55

0.45

Ser

73

.

.

B

.

.

.

.

0.03

.

*

F

0.90

0.61

Ile

74

.

.

B

.

.

T

.

0.34

.

*

F

0.85

0.49

Pro

75

.

.

B

.

.

T

.

−0.12

.

*

F

1.00

1.04

Ser

76

.

.

.

.

.

T

C

−0.12

.

.

F

1.05

0.64

Ser

77

.

.

.

.

.

T

C

0.69

*

.

F

1.20

1.57

Glu

78

A

A

.

.

.

.

.

0.10

*

.

F

0.90

1.64

Leu

79

.

A

B

.

.

.

.

0.78

*

.

F

0.71

0.86

Glu

80

.

A

.

.

T

.

.

0.69

*

.

F

1.37

0.99

Asn

81

.

A

.

.

.

.

C

0.99

.

.

F

1.43

0.76

Ile

82

.

.

.

.

.

.

C

1.29

*

.

F

2.04

1.61

Pro

83

.

.

.

.

.

.

C

1.29

.

.

F

2.60

1.49

Ser

84

.

.

.

.

T

T

.

1.86

*

.

F

2.44

1.61

Glu

85

A

.

.

.

.

T

.

1.16

*

.

F

1.18

3.59

Asn

86

A

.

.

.

.

T

.

1.16

.

.

F

0.92

2.01

Gln

87

.

.

.

.

T

T

.

1.74

*

.

F

1.06

2.60

Tyr

88

.

.

B

B

.

.

.

1.37

*

.

.

−0.15

2.01

Phe

89

.

.

B

B

.

.

.

0.78

.

.

.

−0.45

1.26

Gln

90

.

.

B

B

.

.

.

0.49

.

.

.

−0.60

0.51

Ser

91

.

.

B

B

.

.

.

0.19

*

.

.

−0.60

0.34

Ala

92

.

.

B

B

.

.

.

−0.16

*

.

.

−0.60

0.53

Ile

93

.

.

B

B

.

.

.

−0.61

.

.

.

−0.60

0.30

Trp

94

A

.

.

.

.

T

.

−0.80

*

*

.

−0.20

0.20

Ser

95

A

.

.

.

.

T

.

−0.76

*

*

.

−0.20

0.14

Gly

96

A

.

.

.

.

T

.

−1.31

*

*

.

−0.20

0.39

Phe

97

A

.

.

.

.

T

.

−0.68

*

*

.

−0.20

0.27

Ile

98

.

A

B

B

.

.

.

0.26

*

*

.

0.30

0.41

Lys

99

A

A

.

B

.

.

.

0.24

.

*

.

0.60

0.83

Val

100

.

A

.

B

.

.

C

0.54

.

*

F

1.70

1.28

Lys

101

.

A

.

B

.

.

C

0.89

.

*

F

2.00

3.05

Lys

102

.

A

.

.

.

.

C

1.34

.

.

F

2.30

2.64

Ser

103

.

.

.

.

.

T

C

1.92

.

*

F

3.00

5.57

Asp

104

A

.

.

.

.

T

.

1.18

.

.

F

2.50

4.02

Glu

105

A

.

.

.

.

T

.

1.44

.

.

F

2.20

1.74

Tyr

106

.

.

B

.

.

T

.

1.09

.

.

.

1.45

1.31

Thr

107

A

.

.

B

.

.

.

0.74

.

.

.

0.15

1.13

Phe

108

A

.

.

B

.

.

.

0.46

.

.

.

−0.30

0.88

Ala

109

A

.

.

B

.

.

.

0.46

.

.

.

−0.60

0.57

Thr

110

A

.

.

B

.

.

.

0.46

.

.

F

0.06

0.65

Ser

111

A

.

.

.

.

T

.

0.67

*

.

F

0.82

1.22

Ala

112

A

.

.

.

.

T

.

0.12

*

.

F

1.63

1.64

Asp

113

A

.

.

.

.

T

.

0.51

*

.

F

1.69

0.84

Asn

114

.

.

.

.

.

T

C

0.50

*

.

F

2.10

0.91

His

115

.

.

.

B

.

.

C

0.52

*

.

.

0.74

0.89

Val

116

.

.

B

B

.

.

.

−0.03

*

*

.

0.03

0.56

Thr

117

.

.

B

B

.

.

.

0.56

*

.

.

−0.18

0.26

Met

118

.

.

B

B

.

.

.

0.56

.

.

.

−0.39

0.32

Trp

119

A

.

.

B

.

.

.

0.56

.

.

.

−0.30

0.71

Val

120

A

.

.

.

.

T

.

0.59

.

.

.

0.10

0.86

Asp

121

A

.

.

.

.

T

.

0.59

.

.

F

1.00

1.50

Asp

122

A

.

.

.

.

T

.

0.01

.

.

F

1.00

1.06

Gln

123

A

.

.

.

.

T

.

0.61

*

.

F

1.15

1.00

Glu

124

A

A

.

B

.

.

.

0.94

*

.

F

0.75

0.96

Val

125

A

A

.

B

.

.

.

1.21

.

.

.

0.75

1.15

Ile

126

A

A

.

B

.

.

.

0.91

*

.

.

0.30

0.67

Asn

127

A

A

.

B

.

.

.

0.91

*

.

.

0.60

0.52

Lys

128

A

A

.

.

.

.

.

0.61

*

.

F

0.60

1.13

Ala

129

A

A

.

.

.

.

.

0.61

*

.

F

1.50

2.15

Ser

130

.

A

.

.

.

.

C

1.51

*

.

F

2.30

2.15

Asn

131

.

.

.

.

.

T

C

1.51

.

*

F

3.00

2.15

Ser

132

.

.

.

.

.

T

C

1.62

.

*

F

2.40

1.49

Asn

133

A

.

.

.

.

T

.

0.77

.

*

F

2.20

2.18

Lys

134

.

.

B

.

.

T

.

1.36

.

*

F

1.60

1.12

Ile

135

.

A

B

.

.

.

.

1.70

.

*

F

1.20

1.45

Arg

136

.

A

B

.

.

.

.

1.36

.

*

.

0.75

1.80

Leu

137

.

A

B

.

.

.

.

1.77

.

*

F

0.75

0.89

Glu

138

A

A

.

.

.

.

.

0.96

.

*

F

0.90

2.49

Lys

139

A

A

.

.

.

.

.

0.67

.

*

F

0.90

1.05

Gly

140

A

.

.

B

.

.

.

1.56

*

.

F

0.60

1.99

Arg

141

A

.

.

B

.

.

.

0.56

.

.

F

0.90

1.99

Leu

142

A

.

.

B

.

.

.

1.41

.

*

.

0.30

0.70

Tyr

143

A

.

.

B

.

.

.

0.52

.

*

.

0.45

1.41

Gln

144

.

.

B

B

.

.

.

0.48

.

*

.

−0.30

0.50

Ile

145

.

.

B

B

.

.

.

0.58

.

*

.

−0.45

1.06

Lys

146

.

.

B

B

.

.

.

0.47

*

*

.

−0.45

1.06

Ile

147

.

.

B

B

.

.

.

1.39

*

*

.

−0.15

1.06

Gln

148

.

.

B

B

.

.

.

1.63

.

*

.

0.45

2.96

Tyr

149

.

.

B

.

.

.

.

1.63

.

*

.

0.95

2.56

Gln

150

.

.

B

.

.

.

.

2.31

.

*

F

0.80

5.88

Arg

151

.

.

.

.

T

.

.

1.96

.

.

F

1.84

5.25

Glu

152

.

.

.

.

.

.

C

2.84

.

.

F

1.98

4.83

Asn

153

.

.

.

.

.

T

C

2.89

.

.

F

2.52

4.83

Pro

154

.

.

.

.

.

T

C

2.79

.

.

F

2.86

4.93

Thr

155

.

.

.

.

T

T

.

1.98

.

.

F

3.40

2.82

Glu

156

A

.

.

.

.

T

.

1.87

.

*

F

2.66

1.45

Lys

157

A

A

.

.

.

.

.

1.17

.

*

F

1.92

1.56

Gly

158

A

A

.

.

.

.

.

1.21

.

*

F

1.43

0.94

Leu

159

A

A

.

.

.

.

.

0.61

.

*

.

1.09

1.08

Asp

160

A

A

.

.

.

.

.

0.68

.

*

.

0.30

0.45

Phe

161

.

.

B

B

.

.

.

0.39

.

*

.

−0.30

0.71

Lys

162

.

.

B

B

.

.

.

0.03

.

*

.

−0.60

0.90

Leu

163

.

.

B

B

.

.

.

0.38

.

*

.

−0.60

0.78

Tyr

164

.

.

B

B

.

.

.

0.89

.

*

.

−0.45

1.50

Trp

165

A

.

.

B

.

.

.

0.89

.

*

.

0.15

1.01

Thr

166

A

.

.

B

.

.

.

1.59

.

*

F

0.30

2.11

Asp

167

A

.

.

B

.

.

.

1.59

.

.

F

0.90

2.17

Ser

168

A

.

.

.

.

T

.

2.44

.

.

F

2.20

4.12

Gln

169

.

.

.

.

.

T

C

2.69

.

.

F

3.00

5.71

Asn

170

.

.

.

.

.

T

C

2.12

.

.

F

2.70

5.93

Lys

171

.

.

.

.

.

T

C

1.54

.

.

F

2.40

3.28

Lys

172

.

A

B

.

.

.

.

1.24

.

.

F

1.50

1.33

Glu

173

.

A

B

.

.

.

.

1.24

.

.

F

1.20

1.11

Val

174

.

A

B

.

.

.

.

1.24

.

.

F

1.03

0.74

Ile

175

.

A

B

.

.

.

.

1.24

.

.

F

1.31

0.62

Ser

176

.

.

B

.

.

T

.

0.39

.

.

F

1.99

0.58

Ser

177

.

.

B

.

.

T

.

0.34

.

.

F

1.37

0.64

Asp

178

.

.

.

.

T

T

.

−0.47

.

.

F

2.80

1.58

Asn

179

.

.

.

.

.

T

C

0.18

.

.

F

2.17

0.97

Leu

180

A

A

.

.

.

.

.

1.07

.

.

.

1.29

1.12

Gln

181

A

A

.

.

.

.

.

0.56

.

.

.

1.01

1.16

Leu

182

A

A

.

.

.

.

.

0.90

.

.

.

−0.02

0.60

Pro

183

A

A

.

.

.

.

.

0.90

.

.

F

0.60

1.45

Glu

184

A

A

.

.

.

.

.

0.94

.

*

F

0.60

1.45

Leu

185

A

A

.

.

.

.

.

1.46

.

.

F

0.90

3.51

Lys

186

A

A

.

.

.

.

.

1.16

*

.

F

0.90

3.04

Gln

187

A

A

.

.

.

.

.

1.97

*

.

F

1.24

2.35

Lys

188

A

A

.

.

.

.

.

1.88

.

*

F

1.58

4.59

Ser

189

A

.

.

.

.

T

.

1.99

*

*

F

2.32

3.07

Ser

190

A

.

.

.

.

T

.

2.84

.

*

F

2.66

3.48

Asn

191

.

.

.

.

T

T

.

2.84

.

.

F

3.40

3.48

Ser

192

.

.

.

.

T

T

.

2.96

.

.

F

3.06

5.19

Arg

193

.

.

.

.

T

.

.

2.61

.

.

F

2.52

7.58

Lys

194

.

.

.

.

T

.

.

2.60

.

.

F

2.18

6.32

Lys

195

.

.

.

.

T

.

.

2.60

.

.

F

1.84

6.80

Arg

196

.

.

B

.

.

.

.

2.01

.

.

F

1.10

4.65

Ser

197

.

.

B

.

.

.

.

1.97

.

.

F

1.10

2.35

Thr

198

.

.

B

.

.

.

.

1.64

.

.

F

1.10

1.16

Ser

199

.

.

.

.

T

T

.

1.29

.

.

F

1.25

0.92

Ala

200

.

.

.

.

.

T

C

0.39

*

.

F

0.71

0.99

Gly

201

.

.

.

.

.

T

C

0.07

*

.

F

0.97

0.51

Pro

202

.

.

B

.

.

T

.

0.37

*

.

F

1.03

0.59

Thr

203

.

.

B

.

.

.

.

0.79

.

.

F

1.69

0.97

Val

204

.

.

B

.

.

T

.

1.09

.

.

F

2.60

1.92

Pro

205

.

.

B

.

.

T

.

1.68

*

.

F

2.34

2.07

Asp

206

.

.

B

.

.

T

.

2.02

*

.

F

2.42

2.31

Arg

207

.

.

B

.

.

T

.

1.89

*

.

F

2.50

5.20

Asp

208

.

.

.

.

T

T

.

1.31

*

.

F

2.98

3.33

Asn

209

.

.

.

.

T

T

.

1.96

*

.

F

3.06

1.40

Asp

210

.

.

.

.

T

T

.

2.17

*

.

F

3.40

1.10

Gly

211

.

.

.

.

.

T

C

1.87

*

.

F

2.86

1.10

Ile

212

.

.

.

.

.

T

C

0.94

*

.

F

2.37

0.92

Pro

213

.

.

.

.

.

T

C

0.94

*

.

F

1.73

0.45

Asp

214

.

.

.

.

.

T

C

0.09

*

*

F

1.39

0.79

Ser

215

.

.

B

.

.

T

.

0.09

*

*

F

0.85

0.84

Leu

216

.

A

B

.

.

.

.

0.09

.

*

.

0.60

0.94

Glu

217

.

A

B

.

.

.

.

0.73

.

*

.

0.60

0.56

Val

218

A

A

.

.

.

.

.

0.63

.

.

.

0.30

0.65

Glu

219

A

A

.

.

.

.

.

−0.22

.

.

.

0.45

1.14

Gly

220

A

A

.

.

.

.

.

0.08

.

*

.

0.30

0.49

Tyr

221

A

.

.

B

.

.

.

0.03

.

*

.

0.45

1.10

Thr

222

A

.

.

B

.

.

.

0.08

.

*

.

0.30

0.47

Val

223

A

.

.

B

.

.

.

0.93

.

*

.

0.56

0.95

Asp

224

A

.

.

B

.

.

.

0.98

.

.

.

0.82

0.98

Val

225

A

.

.

.

.

.

.

1.43

.

*

F

1.88

1.36

Lys

226

A

.

.

.

.

.

.

1.37

.

*

F

2.14

3.58

Asn

227

.

.

B

.

.

T

.

0.98

.

*

F

2.60

3.09

Lys

228

.

.

B

.

.

T

.

1.02

.

*

F

2.34

3.61

Arg

229

.

.

B

.

.

T

.

0.72

.

.

F

2.08

1.49

Thr

230

.

.

B

.

.

T

.

1.37

*

.

F

1.52

1.24

Phe

231

.

.

B

.

.

.

.

1.03

*

.

F

0.91

0.96

Leu

232

.

.

B

.

.

.

.

0.14

.

.

.

−0.40

0.51

Ser

233

.

.

.

.

.

T

C

−0.20

.

.

.

0.00

0.25

Pro

234

.

.

.

.

T

T

.

−0.31

*

.

.

0.20

0.39

Trp

235

.

.

.

.

T

T

.

−0.89

*

.

.

0.20

0.75

Ile

236

A

.

.

.

.

T

.

−0.22

*

.

.

−0.20

0.39

Ser

237

A

.

.

B

.

.

.

0.59

*

.

.

−0.60

0.35

Asn

238

A

A

.

B

.

.

.

0.93

.

.

.

−0.60

0.57

Ile

239

A

A

.

B

.

.

.

1.19

.

.

.

0.45

1.63

His

240

A

A

.

B

.

.

.

1.13

.

.

.

0.75

2.44

Glu

241

A

A

.

.

.

.

.

1.21

.

.

F

0.90

1.50

Lys

242

A

A

.

.

.

.

.

1.20

.

.

F

0.90

1.76

Lys

243

A

A

.

.

.

.

.

1.24

.

.

F

0.90

1.87

Gly

244

A

A

.

.

.

.

.

1.89

.

*

F

0.90

2.16

Leu

245

A

.

.

.

.

.

.

1.97

*

.

F

1.44

1.69

Thr

246

A

.

.

.

.

T

.

1.67

*

.

F

1.98

1.69

Lys

247

.

.

B

.

.

T

.

1.32

*

.

F

2.02

2.29

Tyr

248

.

.

B

.

.

T

.

1.07

*

*

F

2.36

3.72

Lys

249

.

.

.

.

T

T

.

1.41

*

.

F

3.40

3.99

Ser

250

.

.

.

.

.

.

C

2.27

*

.

F

2.66

3.45

Ser

251

.

.

.

.

.

T

C

2.29

*

*

F

2.52

4.41

Pro

252

.

.

.

.

.

T

C

1.94

*

*

F

2.18

2.32

Glu

253

.

.

.

.

T

T

.

1.88

*

.

F

2.04

2.32

Lys

254

.

.

.

.

T

T

.

1.24

*

.

F

1.40

2.50

Trp

255

.

.

.

.

T

.

.

1.24

.

.

F

1.20

1.63

Ser

256

.

.

B

.

.

.

.

1.54

.

.

F

0.80

1.26

Thr

257

.

.

B

.

.

.

.

1.54

*

.

F

1.10

1.05

Ala

258

.

.

.

.

T

.

.

1.30

*

.

F

1.20

1.55

Ser

259

.

.

.

.

.

.

C

0.96

*

.

F

1.90

1.81

Asp

260

.

.

.

.

.

T

C

1.24

*

.

F

2.40

1.68

Pro

261

.

.

.

.

.

T

C

0.84

*

.

F

3.00

2.78

Tyr

262

.

.

.

.

T

T

.

1.16

*

.

F

2.60

1.80

Ser

263

.

.

.

.

.

T

C

1.79

*

.

F

2.40

1.86

Asp

264

A

A

.

.

.

.

.

1.23

*

.

F

1.50

2.41

Phe

265

A

A

.

.

.

.

.

0.92

*

.

F

0.90

1.14

Glu

266

A

A

.

.

.

.

.

0.79

*

*

F

0.90

1.23

Lys

267

A

A

.

.

.

.

.

1.14

*

*

F

0.75

0.73

Val

268

A

A

.

.

.

.

.

0.56

*

*

F

0.90

1.65

Thr

269

A

.

.

B

.

.

.

0.56

*

*

F

0.75

0.67

Gly

270

A

.

.

B

.

.

.

1.30

*

*

F

0.75

0.56

Arg

271

A

.

.

B

.

.

.

1.30

*

*

F

0.90

1.50

Ile

272

.

.

B

B

.

.

.

0.40

*

*

F

1.20

1.67

Asp

273

.

.

.

.

T

T

.

0.96

*

*

F

2.30

1.25

Lys

274

.

.

.

.

.

T

C

1.06

*

*

F

2.25

0.86

Asn

275

.

.

.

.

.

T

C

1.40

*

*

F

2.40

1.89

Val

276

.

.

.

.

.

T

C

0.70

*

*

F

3.00

1.96

Ser

277

.

.

.

.

.

T

C

1.70

*

*

F

2.55

0.99

Pro

278

.

.

.

.

.

T

C

1.67

*

*

F

2.40

1.21

Glu

279

A

.

.

.

.

T

.

1.41

*

*

F

1.90

2.21

Ala

280

A

.

.

.

.

T

.

0.60

*

*

F

1.60

2.56

Arg

281

A

.

.

.

.

.

.

0.60

.

*

.

0.65

1.36

His

282

.

.

B

B

.

.

.

0.31

.

*

.

0.30

0.58

Pro

283

.

.

B

B

.

.

.

−0.07

.

*

.

−0.30

0.58

Leu

284

A

.

.

B

.

.

.

−0.31

.

*

.

−0.30

0.30

Val

285

A

.

.

B

.

.

.

0.07

*

*

.

−0.60

0.35

Ala

286

A

.

.

B

.

.

.

−0.93

*

.

.

−0.60

0.35

Ala

287

.

.

B

B

.

.

.

−1.76

.

.

.

−0.60

0.29

Tyr

288

.

.

B

B

.

.

.

−1.58

.

.

.

−0.60

0.29

Pro

289

.

.

B

B

.

.

.

−1.62

.

.

.

−0.60

0.40

Ile

290

.

.

B

B

.

.

.

−0.77

.

.

.

−0.60

0.29

Val

291

.

.

B

B

.

.

.

−0.78

.

*

.

−0.60

0.31

His

292

.

.

B

B

.

.

.

−0.19

.

*

.

−0.60

0.20

Val

293

.

.

B

B

.

.

.

0.06

.

.

.

−0.30

0.49

Asp

294

A

.

.

B

.

.

.

−0.62

.

.

.

0.45

1.07

Met

295

A

A

.

.

.

.

.

−0.62

.

.

.

0.30

0.55

Glu

296

A

A

.

.

.

.

.

−0.58

.

*

.

−0.30

0.52

Asn

297

A

A

.

.

.

.

.

−0.84

.

.

.

−0.30

0.26

Ile

298

A

A

.

.

.

.

.

0.06

.

.

.

−0.60

0.35

Ile

299

A

A

.

.

.

.

.

0.06

.

.

.

0.30

0.40

Leu

300

A

A

.

.

.

.

.

0.66

.

.

.

0.04

0.40

Ser

301

A

A

.

.

.

.

.

0.66

.

.

F

1.13

0.99

Lys

302

A

A

.

.

.

.

.

0.66

.

.

F

1.92

2.36

Asn

303

.

.

.

.

.

T

C

1.24

.

.

F

2.86

4.95

Glu

304

.

.

.

.

T

T

.

1.82

*

.

F

3.40

4.95

Asp

305

.

.

.

.

T

T

.

2.63

*

.

F

3.06

3.57

Gln

306

.

.

.

.

T

T

.

2.93

.

.

F

2.72

3.85

Ser

307

.

.

.

.

T

.

.

2.58

.

.

F

2.18

3.57

Thr

308

.

.

B

.

.

.

.

2.58

.

.

F

1.42

3.09

Gln

309

.

.

.

.

T

.

.

2.28

.

.

F

1.76

2.98

Asn

310

.

.

.

.

.

T

C

2.28

.

.

F

2.04

2.98

Thr

311

.

.

.

.

.

T

C

1.97

*

*

F

2.32

3.57

Asp

312

.

.

.

.

T

T

.

2.38

.

*

F

2.80

2.98

Ser

313

.

.

B

.

.

T

.

2.38

*

.

F

2.42

3.62

Gln

314

.

.

B

B

.

.

.

1.49

*

.

F

1.74

3.62

Thr

315

.

.

B

B

.

.

.

1.19

*

*

F

1.46

1.52

Arg

316

.

.

B

B

.

.

.

1.54

*

.

F

0.88

1.52

Thr

317

.

.

B

B

.

.

.

1.54

*

.

F

0.90

1.76

Ile

318

.

.

B

B

.

.

.

1.53

*

.

F

0.90

1.96

Ser

319

.

.

B

.

.

T

.

1.23

*

.

F

1.60

1.44

Lys

320

.

.

.

.

T

T

.

1.23

*

.

F

2.00

1.34

Asn

321

.

.

.

.

.

T

C

0.82

*

.

F

2.10

2.76

Thr

322

.

.

.

.

.

T

C

1.24

.

.

F

2.40

2.76

Ser

323

.

.

.

.

.

T

C

1.82

.

.

F

3.00

2.70

Thr

324

.

.

.

.

.

T

C

2.09

.

.

F

2.40

2.42

Ser

325

.

.

.

.

.

T

C

1.73

.

.

F

2.36

2.29

Arg

326

.

.

.

.

.

T

C

1.43

.

.

F

2.32

2.46

Thr

327

.

.

.

.

.

.

C

1.74

.

.

F

2.08

2.29

His

328

.

.

.

.

.

T

C

1.19

*

.

F

2.54

2.95

Thr

329

.

.

B

.

.

T

.

1.47

.

.

F

2.60

1.12

Ser

330

.

.

B

.

.

T

.

1.42

.

*

F

2.04

1.06

Glu

331

.

.

B

.

.

T

.

1.31

.

*

F

1.63

0.77

Val

332

.

.

.

.

.

.

C

1.03

.

*

F

1.37

0.86

His

333

.

.

.

.

.

T

C

1.07

.

*

F

1.31

0.64

Gly

334

.

.

.

.

.

T

C

0.52

.

*

.

1.20

0.64

Asn

335

A

.

.

.

.

T

.

0.79

.

*

.

0.10

0.64

Ala

336

A

.

.

.

.

T

.

0.20

.

*

.

0.70

0.64

Glu

337

A

A

.

.

.

.

.

0.76

.

*

.

0.30

0.66

Val

338

A

A

.

.

.

.

.

0.09

.

*

.

0.30

0.55

His

339

A

A

.

.

.

.

.

−0.27

.

*

.

−0.30

0.47

Ala

340

A

A

.

.

.

.

.

−0.27

.

*

.

−0.60

0.24

Ser

341

.

A

B

.

.

.

.

−0.57

*

*

.

−0.60

0.53

Phe

342

.

A

B

.

.

.

.

−0.91

*

.

.

−0.60

0.27

Phe

343

.

A

B

.

.

.

.

−0.40

.

.

.

−0.60

0.27

Asp

344

.

.

.

.

T

T

.

−0.67

*

*

.

0.20

0.20

Ile

345

.

.

.

.

T

T

.

−0.93

*

*

.

0.20

0.30

Gly

346

.

.

.

.

T

T

.

−0.93

*

*

F

0.65

0.26

Gly

347

.

.

.

.

.

T

C

−0.82

*

*

F

1.05

0.21

Ser

348

.

.

.

.

.

.

C

−0.47

*

*

F

−0.05

0.30

Val

349

.

.

B

.

.

.

.

−1.17

*

*

F

0.05

0.30

Ser

350

.

.

B

.

.

T

.

−0.58

*

*

.

−0.20

0.26

Ala

351

.

.

B

.

.

T

.

−0.23

.

.

.

−0.08

0.26

Gly

352

.

.

B

.

.

T

.

−0.19

.

.

.

0.04

0.57

Phe

353

.

.

B

.

.

T

.

0.11

.

.

F

0.61

0.57

Ser

354

.

.

.

.

.

.

C

0.67

.

.

F

0.73

0.91

Asn

355

.

.

.

.

.

T

C

0.67

.

.

F

1.20

1.24

Ser

356

.

.

.

.

.

T

C

0.94

.

.

F

1.08

1.91

Asn

357

.

.

.

.

.

T

C

0.43

.

.

F

1.56

2.06

Ser

358

.

.

.

.

.

T

C

0.54

.

.

F

0.69

0.95

Ser

359

.

.

.

B

.

.

C

−0.04

.

.

F

0.17

0.72

Thr

360

.

.

B

B

.

.

.

−0.04

.

.

F

−0.45

0.31

Val

361

.

.

B

B

.

.

.

0.22

.

.

.

−0.30

0.39

Ala

362

.

.

B

B

.

.

.

−0.08

.

*

.

−0.30

0.40

Ile

363

.

.

B

B

.

.

.

−0.59

.

*

.

−0.30

0.37

Asp

364

.

.

B

B

.

.

.

−0.59

.

*

.

−0.30

0.41

His

365

.

.

B

.

.

.

.

−1.09

.

*

.

−0.10

0.54

Ser

366

A

A

.

.

.

.

.

−0.82

.

*

.

−0.30

0.64

Leu

367

.

A

B

.

.

.

.

−0.58

*

*

.

−0.30

0.38

Ser

368

.

A

.

.

.

.

C

0.31

*

*

.

−0.40

0.28

Leu

369

.

A

.

.

.

.

C

0.42

.

.

.

−0.10

0.36

Ala

370

A

A

.

.

.

.

.

0.14

*

.

.

0.30

0.86

Gly

371

A

A

.

.

.

.

.

0.16

*

.

F

0.75

0.93

Glu

372

A

A

.

.

.

.

.

0.38

*

.

F

0.00

1.18

Arg

373

A

A

.

.

.

.

.

0.68

.

.

F

0.60

1.18

Thr

374

A

A

.

.

.

.

.

1.18

*

.

F

0.90

2.06

Trp

375

A

A

.

.

.

.

.

1.17

*

.

.

0.75

1.72

Ala

376

A

A

.

.

.

.

.

1.17

*

.

.

0.30

0.87

Glu

377

A

A

.

.

.

.

.

0.36

.

.

.

−0.60

0.60

Thr

378

A

A

.

.

.

.

.

0.24

.

.

.

−0.60

0.47

Met

379

A

A

.

.

.

.

.

0.24

.

.

.

−0.30

0.74

Gly

380

.

A

.

.

.

.

C

−0.06

.

.

.

−0.10

0.62

Leu

381

.

A

.

.

.

.

C

0.53

.

.

.

−0.40

0.43

Asn

382

A

A

.

.

.

.

.

0.22

.

.

F

−0.15

0.73

Thr

383

A

A

.

.

.

.

.

−0.06

*

.

F

0.60

1.07

Ala

384

A

A

.

.

.

.

.

0.66

.

*

F

0.00

1.31

Asp

385

A

A

.

.

.

.

.

0.19

.

*

F

0.90

1.59

Thr

386

A

A

.

.

.

.

.

1.00

.

*

F

0.45

0.91

Ala

387

A

A

.

.

.

.

.

0.41

*

*

F

0.60

1.45

Arg

388

A

A

.

.

.

.

.

0.72

*

*

.

0.30

0.88

Leu

389

A

A

.

.

.

.

.

0.42

.

*

.

0.30

0.98

Asn

390

A

.

.

.

.

T

.

0.53

*

*

.

0.10

0.68

Ala

391

.

.

B

.

.

T

.

0.60

*

*

.

0.70

0.68

Asn

392

.

.

B

.

.

T

.

0.33

*

*

.

−0.05

1.29

Ile

393

.

.

B

.

.

T

.

0.22

*

*

.

0.10

0.59

Arg

394

.

.

B

B

.

.

.

0.72

*

*

.

−0.30

0.95

Tyr

395

.

.

B

B

.

.

.

0.38

*

*

.

−0.30

0.85

Val

396

.

.

B

B

.

.

.

0.66

*

*

.

−0.15

1.20

Asn

397

.

.

B

.

.

T

.

0.07

*

*

F

0.25

0.88

Thr

398

.

.

B

.

.

T

.

0.74

*

*

F

−0.05

0.57

Gly

399

.

.

.

.

T

T

.

−0.26

*

.

F

0.80

1.19

Thr

400

.

.

B

.

.

T

.

−0.26

*

.

F

−0.05

0.52

Ala

401

.

.

B

B

.

.

.

0.60

*

.

F

−0.45

0.56

Pro

402

.

.

B

B

.

.

.

−0.26

*

.

.

−0.60

0.91

Ile

403

.

.

B

B

.

.

.

−0.76

*

.

.

−0.60

0.47

Tyr

404

.

.

B

B

.

.

.

−0.62

*

.

.

−0.60

0.38

Asn

405

.

.

B

B

.

.

.

−0.62

.

.

.

−0.60

0.38

Val

406

.

.

B

B

.

.

.

−0.34

.

.

.

−0.60

0.79

Leu

407

.

.

B

B

.

.

.

−0.43

.

.

.

−0.60

0.73

Pro

408

.

.

B

.

.

T

.

−0.36

.

.

F

−0.05

0.61

Thr

409

.

.

B

.

.

T

.

−0.97

.

.

F

−0.05

0.67

Thr

410

.

.

B

.

.

T

.

−1.78

.

.

F

−0.05

0.61

Ser

411

.

.

B

.

.

T

.

−1.27

.

.

F

−0.05

0.32

Leu

412

.

.

B

B

.

.

.

−0.41

.

.

.

−0.60

0.22

Val

413

.

.

B

B

.

.

.

−0.20

.

.

.

−0.30

0.31

Leu

414

.

.

B

B

.

.

.

0.11

.

.

.

−0.30

0.37

Gly

415

.

.

.

.

T

T

.

0.11

.

.

F

0.65

0.77

Lys

416

A

.

.

.

.

T

.

−0.40

.

.

F

0.40

1.50

Asn

417

A

.

.

.

.

T

.

−0.18

.

.

F

0.40

1.50

Gln

418

A

.

.

.

.

T

.

0.37

.

.

F

1.00

1.54

Thr

419

A

.

.

B

.

.

.

0.29

.

*

F

0.60

1.11

Leu

420

A

.

.

B

.

.

.

0.68

.

*

F

−0.45

0.48

Ala

421

A

.

.

B

.

.

.

0.04

.

*

.

−0.30

0.56

Thr

422

A

.

.

B

.

.

.

0.09

.

*

.

−0.30

0.39

Ile

423

A

.

.

B

.

.

.

0.09

.

*

.

0.30

0.95

Lys

424

A

.

.

B

.

.

.

0.40

.

*

F

0.90

1.62

Ala

425

A

A

.

.

.

.

.

1.21

.

*

F

0.90

1.81

Lys

426

A

A

.

.

.

.

.

0.99

.

*

F

0.90

4.47

Glu

427

A

A

.

.

.

.

.

1.00

*

*

F

0.90

1.84

Asn

428

A

A

.

.

.

.

.

1.89

*

*

F

0.90

2.44

Gln

429

A

A

.

B

.

.

.

0.96

*

.

F

0.90

2.12

Leu

430

A

A

.

B

.

.

.

0.73

.

.

F

0.45

0.86

Ser

431

.

A

B

B

.

.

.

0.10

.

.

F

−0.45

0.44

Gln

432

.

A

B

B

.

.

.

−0.11

.

.

.

−0.60

0.26

Ile

433

.

A

B

B

.

.

.

−0.11

*

.

.

−0.60

0.48

Leu

434

.

A

B

B

.

.

.

−0.11

.

.

.

−0.60

0.58

Ala

435

.

.

B

.

.

T

.

0.46

.

.

.

−0.20

0.54

Pro

436

.

.

B

.

.

T

.

0.51

.

.

F

0.10

1.20

Asn

437

.

.

.

.

T

T

.

0.30

.

.

F

0.50

2.27

Asn

438

.

.

.

.

T

T

.

0.89

.

.

F

0.50

3.48

Tyr

439

.

.

.

.

T

.

.

1.74

.

.

F

0.30

3.02

Tyr

440

.

.

B

.

.

T

.

2.33

.

.

F

0.40

3.75

Pro

441

.

.

.

.

T

T

.

1.73

.

.

F

0.80

3.75

Ser

442

.

.

.

.

T

T

.

1.14

.

.

F

0.50

1.97

Lys

443

.

.

B

.

.

T

.

0.93

.

.

F

0.40

1.27

Asn

444

.

A

B

.

.

.

.

0.29

*

.

F

0.60

1.27

Leu

445

.

A

B

.

.

.

.

−0.06

.

.

.

−0.30

0.67

Ala

446

.

A

B

.

.

.

.

−0.66

*

.

.

−0.30

0.34

Pro

447

.

A

B

.

.

.

.

−0.36

*

.

.

−0.60

0.17

Ile

448

.

A

B

.

.

.

.

−0.99

*

*

.

−0.60

0.34

Ala

449

A

A

.

.

.

.

.

−0.99

*

.

.

−0.60

0.34

Leu

450

.

A

B

.

.

.

.

−0.18

*

*

.

−0.60

0.38

Asn

451

.

A

B

.

.

.

.

0.41

*

*

.

0.04

0.90

Ala

452

A

A

.

.

.

.

.

−0.08

*

*

F

1.58

1.48

Gln

453

A

A

.

.

.

.

.

0.51

*

*

F

1.62

1.56

Asp

454

.

.

.

.

T

T

.

0.80

*

*

F

3.06

1.30

Asp

455

.

.

.

.

T

T

.

1.30

.

*

F

3.40

1.72

Phe

456

.

.

.

.

T

T

.

1.09

.

*

F

3.06

1.43

Ser

457

.

.

B

.

.

T

.

0.79

.

.

F

2.32

1.33

Ser

458

.

.

B

B

.

.

.

0.48

.

.

F

0.53

0.56

Thr

459

.

.

.

B

.

.

C

−0.12

.

.

F

0.09

0.93

Pro

460

.

.

.

B

.

.

C

−0.12

.

.

F

−0.25

0.69

Ile

461

.

.

.

B

T

.

.

0.33

.

*

F

−0.05

0.82

Thr

462

.

.

B

B

.

.

.

0.63

.

.

.

−0.60

0.89

Met

463

.

.

B

B

.

.

.

0.93

.

.

.

−0.60

0.93

Asn

464

.

.

B

.

.

T

.

0.54

*

.

.

−0.05

2.30

Tyr

465

.

.

B

.

.

T

.

−0.06

*

.

.

−0.05

1.38

Asn

466

.

.

.

.

.

T

C

0.83

*

*

.

0.15

1.15

Gln

467

A

.

.

.

.

T

.

0.33

.

*

.

−0.05

1.24

Phe

468

A

A

.

.

.

.

.

0.93

.

.

.

−0.60

0.65

Leu

469

A

A

.

.

.

.

.

0.98

*

.

.

0.30

0.70

Glu

470

A

A

.

.

.

.

.

0.91

.

.

.

0.30

0.81

Leu

471

A

A

.

.

.

.

.

0.96

.

.

.

0.45

1.35

Glu

472

A

A

.

.

.

.

.

0.96

.

.

F

0.90

3.27

Lys

473

A

A

.

.

.

.

.

0.84

.

*

F

0.90

3.27

Thr

474

A

A

.

.

.

.

.

1.77

.

*

F

0.90

3.27

Lys

475

A

A

.

.

.

.

.

0.96

.

*

F

0.90

3.70

Gln

476

A

A

.

.

.

.

.

1.77

.

*

F

0.90

1.53

Leu

477

A

A

.

.

.

.

.

1.46

.

*

.

0.98

1.77

Arg

478

.

A

B

.

.

.

.

1.41

.

*

.

1.21

1.27

Leu

479

.

A

B

.

.

.

.

1.72

*

*

.

1.44

1.23

Asp

480

.

.

B

.

.

T

.

0.82

*

*

F

2.22

2.58

Thr

481

.

.

B

.

.

T

.

0.58

.

*

F

2.30

0.98

Asp

482

.

.

B

.

.

T

.

1.04

.

*

F

1.92

1.86

Gln

483

.

.

B

.

.

T

.

0.93

.

*

F

1.69

1.10

Val

484

.

.

B

B

.

.

.

0.86

.

.

.

0.31

1.23

Tyr

485

.

.

B

B

.

.

.

0.27

*

.

.

−0.07

0.52

Gly

486

.

.

B

B

.

.

.

0.27

*

.

.

−0.60

0.30

Asn

487

.

.

B

B

.

.

.

0.02

.

.

.

−0.60

0.58

Ile

488

.

.

B

B

.

.

.

0.02

.

*

.

−0.60

0.58

Ala

489

.

.

B

B

.

.

.

0.18

.

*

.

−0.60

0.95

Thr

490

.

.

B

B

.

.

.

0.42

.

.

.

−0.60

0.51

Tyr

491

.

.

B

.

.

.

.

0.77

*

.

.

−0.25

1.26

Asn

492

.

.

B

.

.

.

.

0.42

*

.

.

0.39

2.01

Phe

493

.

.

B

.

.

T

.

1.42

.

.

.

0.93

1.38

Glu

494

.

.

.

.

T

T

.

1.16

.

*

F

2.42

1.72

Asn

495

.

.

.

.

T

T

.

1.58

.

*

F

2.61

0.80

Gly

496

.

.

.

.

T

T

.

0.97

.

*

F

3.40

1.80

Arg

497

.

.

B

B

.

.

.

0.97

.

*

F

2.11

0.77

Val

498

.

.

B

B

.

.

.

1.36

.

*

F

1.77

0.80

Arg

499

.

.

B

B

.

.

.

1.01

.

*

.

1.55

1.17

Val

500

.

.

B

B

.

.

.

0.71

.

*

F

1.33

0.59

Asp

501

.

.

B

B

.

.

.

1.06

.

*

F

0.96

1.07

Thr

502

.

.

.

.

.

.

C

0.66

.

*

F

1.63

0.87

Gly

503

.

.

.

.

.

T

C

1.21

.

*

F

1.20

1.24

Ser

504

.

.

.

.

.

T

C

1.10

.

*

F

0.93

0.99

Asn

505

.

.

.

.

.

T

C

1.10

*

.

F

0.96

1.19

Trp

506

.

.

.

.

.

T

C

0.29

*

.

F

0.69

0.89

Ser

507

.

.

.

.

.

.

C

0.39

*

.

F

0.07

0.55

Glu

508

.

.

B

.

.

.

.

0.73

*

.

.

−0.40

0.53

Val

509

.

.

B

B

.

.

.

0.14

*

*

.

−0.60

0.87

Leu

510

.

.

B

B

.

.

.

0.14

*

.

F

−0.15

0.46

Pro

511

.

.

.

B

.

.

C

0.43

*

.

F

0.05

0.46

Gln

512

.

.

.

B

.

.

C

0.42

*

.

F

0.20

1.06

Ile

513

A

.

.

B

.

.

.

0.11

*

.

F

0.00

1.86

Gln

514

A

.

.

B

.

.

.

0.38

*

*

F

0.60

1.74

Glu

515

A

.

.

B

.

.

.

1.30

*

*

F

0.60

1.01

Thr

516

.

.

B

B

.

.

.

0.62

*

.

F

0.60

2.83

Thr

517

.

.

B

B

.

.

.

−0.27

*

*

F

0.60

1.15

Ala

518

.

.

B

B

.

.

.

−0.08

*

*

.

0.30

0.46

Arg

519

.

.

B

B

.

.

.

−0.08

*

*

.

−0.60

0.28

Ile

520

.

.

B

B

.

.

.

−0.42

*

*

.

−0.60

0.31

Ile

521

.

.

B

B

.

.

.

−0.07

*

*

.

−0.36

0.30

Phe

522

.

.

B

B

.

.

.

0.24

*

*

.

0.78

0.31

Asn

523

.

.

.

B

T

.

.

0.02

*

*

F

1.57

0.74

Gly

524

.

.

.

.

T

T

.

−0.09

*

*

F

2.21

0.87

Lys

525

.

.

.

.

.

T

C

−0.01

.

.

F

2.40

1.62

Asp

526

.

.

.

.

.

T

C

0.02

.

.

F

2.01

0.83

Leu

527

.

.

.

.

.

T

C

0.72

*

*

F

1.77

0.62

Asn

528

A

A

.

.

.

.

.

0.83

*

.

.

1.08

0.54

Leu

529

A

A

.

.

.

.

.

1.29

*

.

.

0.84

0.63

Val

530

A

A

.

.

.

.

.

0.36

*

.

.

0.75

1.50

Glu

531

A

A

.

.

.

.

.

−0.23

*

*

.

0.60

0.65

Arg

532

A

A

.

.

.

.

.

−0.01

*

.

.

0.30

0.80

Arg

533

A

A

.

.

.

.

.

−0.87

*

.

.

0.75

1.09

Ile

534

.

A

B

.

.

.

.

−0.06

*

.

.

0.60

0.47

Ala

535

.

A

B

.

.

.

.

0.59

*

.

.

0.30

0.38

Ala

536

.

A

B

.

.

.

.

0.29

.

.

.

−0.30

0.30

Val

537

.

A

B

.

.

.

.

0.18

*

.

.

−0.30

0.58

Asn

538

.

.

.

.

.

T

C

−0.14

.

.

F

1.65

0.96

Pro

539

.

.

.

.

.

T

C

−0.07

.

.

F

2.10

1.46

Ser

540

.

.

.

.

.

T

C

0.52

*

.

F

2.40

1.63

Asp

541

.

.

.

.

.

T

C

0.80

.

.

F

3.00

1.75

Pro

542

.

.

.

.

.

.

C

1.34

.

.

F

2.50

1.64

Leu

543

.

.

B

.

.

.

.

1.39

.

.

F

2.00

1.76

Glu

544

.

.

B

.

.

.

.

1.39

.

.

F

1.70

2.11

Thr

545

A

.

.

.

.

.

.

1.69

.

.

F

1.10

2.11

Thr

546

A

.

.

.

.

.

.

1.09

.

.

F

1.10

4.27

Lys

547

A

.

.

.

.

T

.

0.99

.

*

F

1.30

2.44

Pro

548

A

.

.

.

.

T

.

0.99

.

*

F

1.00

2.44

Asp

549

A

.

.

.

.

T

.

1.03

.

*

F

1.00

1.39

Met

550

A

.

.

.

.

T

.

1.34

*

*

F

1.30

1.39

Thr

551

A

A

.

.

.

.

.

1.07

*

*

.

0.75

1.56

Leu

552

A

A

.

.

.

.

.

0.21

*

*

.

0.60

0.94

Lys

553

A

A

.

.

.

.

.

0.47

*

*

F

0.45

0.79

Glu

554

A

A

.

.

.

.

.

−0.42

*

*

F

0.90

1.09

Ala

555

A

A

.

.

.

.

.

−0.41

*

*

.

0.30

0.93

Leu

556

A

A

.

.

.

.

.

−0.80

*

*

.

0.60

0.47

Lys

557

A

A

.

.

.

.

.

−0.33

*

*

.

−0.30

0.23

Ile

558

A

A

.

.

.

.

.

−1.08

*

*

.

−0.60

0.23

Ala

559

A

A

.

.

.

.

.

−1.08

*

*

.

−0.60

0.24

Phe

560

.

A

B

.

.

.

.

−0.49

*

*

.

−0.60

0.19

Gly

561

.

A

B

.

.

.

.

0.11

.

*

.

−0.32

0.48

Phe

562

.

.

B

.

.

.

.

0.07

.

*

.

0.46

0.73

Asn

563

.

.

.

.

.

.

C

0.61

.

.

F

1.24

1.36

Glu

564

.

.

.

.

.

T

C

1.20

.

.

F

2.32

1.36

Pro

565

.

.

.

.

T

T

.

1.09

.

*

F

2.80

2.53

Asn

566

.

.

.

.

T

T

.

1.43

.

*

F

2.52

1.30

Gly

567

.

.

.

.

T

T

.

1.89

.

*

F

2.24

1.30

Asn

568

.

.

.

.

.

.

C

1.89

.

*

F

0.66

1.31

Leu

569

.

.

B

.

.

.

.

1.54

.

*

.

0.23

1.41

Gln

570

.

.

B

.

.

.

.

1.80

.

*

.

0.15

1.41

Tyr

571

.

.

B

.

.

T

.

1.80

*

*

.

0.85

1.76

Gln

572

.

.

B

.

.

T

.

1.26

*

*

F

1.80

3.56

Gly

573

.

.

B

.

.

T

.

0.94

*

*

F

2.00

1.44

Lys

574

.

.

B

.

.

T

.

1.76

*

*

F

1.80

1.33

Asp

575

.

A

B

.

.

.

.

1.06

*

*

F

1.50

1.33

Ile

576

.

A

B

.

.

.

.

1.30

.

*

F

1.30

1.16

Thr

577

.

A

B

.

.

.

.

0.60

*

*

F

0.95

0.97

Glu

578

.

A

B

.

.

.

.

0.94

*

*

.

0.30

0.50

Phe

579

.

A

B

.

.

.

.

0.20

*

*

.

−0.15

1.15

Asp

580

.

A

.

.

T

.

.

0.20

*

*

.

0.10

0.69

Phe

581

.

A

.

.

T

.

.

1.09

*

*

.

0.70

0.67

Asn

582

.

.

.

.

T

T

.

1.40

.

*

.

0.65

1.34

Phe

583

.

.

.

.

T

T

.

1.09

.

*

F

1.40

1.38

Asp

584

A

.

.

.

.

T

.

1.49

.

*

F

0.40

2.31

Gln

585

A

.

.

.

.

T

.

1.49

.

*

F

1.28

1.92

Gln

586

.

.

.

.

T

.

.

2.19

*

*

F

1.76

3.85

Thr

587

.

.

.

.

.

.

C

1.30

*

*

F

2.14

3.70

Ser

588

.

.

.

.

.

T

C

2.04

*

*

F

1.72

1.50

Gln

589

.

.

.

.

T

T

.

2.04

*

*

F

2.80

1.73

Asn

590

.

.

.

.

T

T

.

2.04

*

*

F

2.52

1.93

Ile

591

.

.

.

.

.

T

C

1.23

*

*

F

2.04

2.49

Lys

592

.

A

.

.

.

.

C

0.96

*

.

F

1.36

1.19

Asn

593

.

A

B

.

.

.

.

1.26

*

.

F

0.13

0.75

Gln

594

.

A

B

.

.

.

.

0.44

*

.

F

0.60

1.84

Leu

595

.

A

B

.

.

.

.

0.44

*

*

.

0.30

0.76

Ala

596

.

A

B

.

.

.

.

0.74

*

*

.

−0.30

0.76

Glu

597

A

A

.

.

.

.

.

0.39

.

.

.

−0.30

0.44

Leu

598

A

A

.

.

.

.

.

0.39

.

.

.

−0.30

0.78

Asn

599

A

A

.

.

.

.

.

−0.50

.

.

.

0.45

1.23

Ala

600

A

.

.

B

.

.

.

0.07

.

.

.

−0.30

0.50

Thr

601

A

.

.

B

.

.

.

0.34

.

.

.

−0.60

0.95

Asn

602

A

.

.

B

.

.

.

−0.51

.

.

.

−0.60

0.85

Ile

603

.

.

B

B

.

.

.

−0.51

.

.

.

−0.60

0.63

Tyr

604

.

.

B

B

.

.

.

−0.51

*

.

.

−0.60

0.36

Thr

605

.

A

B

B

.

.

.

0.12

*

.

.

−0.60

0.37

Val

606

.

A

B

B

.

.

.

−0.46

*

*

.

−0.15

1.06

Leu

607

A

A

.

B

.

.

.

−0.41

*

*

.

−0.30

0.47

Asp

608

A

A

.

B

.

.

.

−0.33

*

*

F

0.45

0.66

Lys

609

A

A

.

B

.

.

.

−0.09

*

*

F

0.45

0.73

Ile

610

A

A

.

B

.

.

.

−0.37

*

*

F

0.90

1.42

Lys

611

A

A

.

B

.

.

.

0.53

*

*

F

0.75

0.86

Leu

612

A

A

.

.

.

.

.

0.74

.

*

F

0.75

0.86

Asn

613

A

A

.

.

.

.

.

0.74

.

*

.

0.45

1.22

Ala

614

A

A

.

.

.

.

.

−0.19

.

*

.

0.30

0.98

Lys

615

A

A

.

.

.

.

.

−0.11

.

*

.

−0.30

0.83

Met

616

A

.

.

B

.

.

.

−1.04

*

*

.

−0.30

0.43

Asn

617

A

.

.

B

.

.

.

−0.12

*

*

.

−0.60

0.30

Ile

618

A

.

.

B

.

.

.

−0.12

.

*

.

−0.30

0.29

Leu

619

A

.

.

B

.

.

.

0.51

.

*

.

−0.30

0.49

Ile

620

A

.

.

B

.

.

.

0.58

.

*

.

0.60

0.61

Arg

621

A

.

.

B

.

.

.

0.48

*

*

.

0.75

1.70

Asp

622

A

.

.

B

.

.

.

0.44

*

*

F

0.90

1.78

Lys

623

.

A

B

.

.

.

.

1.09

*

*

F

0.90

3.46

Arg

624

.

A

B

.

.

.

.

1.90

*

.

.

1.03

2.77

Phe

625

.

A

B

.

.

.

.

2.90

.

.

.

1.31

2.77

His

626

.

A

.

.

T

.

.

2.79

.

.

.

1.99

2.71

Tyr

627

.

A

.

.

T

.

.

2.79

.

*

.

2.27

2.23

Asp

628

.

.

.

.

T

T

.

1.86

*

.

F

2.80

4.13

Arg

629

.

.

.

.

T

T

.

1.16

.

*

F

2.52

2.13

Asn

630

.

.

.

.

T

T

.

1.00

.

.

F

2.24

1.37

Asn

631

.

.

.

.

T

T

.

0.69

.

.

.

1.66

0.61

Ile

632

.

.

B

B

.

.

.

0.34

.

*

.

−0.02

0.31

Ala

633

.

.

B

B

.

.

.

0.34

.

*

.

−0.60

0.19

Val

634

.

.

B

B

.

.

.

0.23

.

*

.

−0.30

0.20

Gly

635

A

.

.

B

.

.

.

−0.07

*

.

.

0.30

0.50

Ala

636

A

A

.

.

.

.

.

−0.92

.

*

F

0.75

0.66

Asp

637

A

A

.

.

.

.

.

−0.89

*

.

F

0.45

0.66

Glu

638

A

A

.

B

.

.

.

−0.26

*

.

F

0.45

0.49

Ser

639

A

A

.

B

.

.

.

0.60

*

.

F

0.75

0.98

Val

640

A

A

.

B

.

.

.

0.36

*

.

F

0.90

1.02

Val

641

A

A

.

B

.

.

.

0.91

*

.

.

0.60

0.59

Lys

642

A

A

.

B

.

.

.

1.02

*

.

.

0.30

0.60

Glu

643

A

A

.

.

.

.

.

1.02

*

.

.

0.75

1.59

Ala

644

A

A

.

.

.

.

.

0.47

*

.

.

0.75

3.70

His

645

A

A

.

.

.

.

.

0.43

*

.

.

0.75

1.37

Arg

646

A

A

.

B

.

.

.

1.29

*

.

.

0.60

0.56

Glu

647

A

A

.

B

.

.

.

0.94

*

.

.

0.54

0.89

Val

648

A

A

.

B

.

.

.

0.64

*

.

.

0.78

0.87

Ile

649

A

A

.

B

.

.

.

0.92

*

.

.

1.02

0.60

Asn

650

.

.

.

.

.

T

C

0.96

*

.

F

2.01

0.50

Ser

651

.

.

.

.

.

T

C

0.50

*

.

F

2.40

1.16

Ser

652

.

.

.

.

.

T

C

−0.31

*

.

F

2.16

1.64

Thr

653

.

.

.

.

.

T

C

−0.27

.

.

F

1.77

0.84

Glu

654

A

A

.

.

.

.

.

−0.19

.

.

F

0.33

0.52

Gly

655

A

A

.

.

.

.

.

−0.19

.

.

F

0.09

0.32

Leu

656

A

A

.

.

.

.

.

−0.78

.

*

.

−0.30

0.35

Leu

657

A

A

.

.

.

.

.

−0.48

*

.

.

−0.60

0.14

Leu

658

A

A

.

.

.

.

.

−0.12

*

.

.

−0.60

0.24

Asn

659

A

A

.

.

.

.

.

−0.12

*

*

.

−0.30

0.59

Ile

660

A

A

.

.

.

.

.

−0.67

*

*

F

0.90

1.19

Asp

661

A

.

.

.

.

T

.

0.26

*

*

F

1.30

1.01

Lys

662

A

.

.

.

.

T

.

1.11

*

*

F

1.30

1.23

Asp

663

A

.

.

.

.

T

.

1.03

*

*

F

1.30

3.51

Ile

664

A

.

.

.

.

T

.

0.22

*

*

F

1.30

1.47

Arg

665

.

.

B

B

.

.

.

0.81

*

*

F

0.75

0.61

Lys

666

.

.

B

B

.

.

.

0.47

*

.

F

0.75

0.49

Ile

667

.

.

B

B

.

.

.

0.18

*

.

.

0.30

0.69

Leu

668

.

.

B

.

.

T

.

−0.71

*

*

.

0.70

0.55

Ser

669

.

.

B

.

.

T

.

−0.68

*

.

.

−0.20

0.19

Gly

670

.

.

B

.

.

T

.

−0.79

*

.

.

−0.20

0.20

Tyr

671

.

.

B

.

.

T

.

−1.72

*

.

.

−0.20

0.43

Ile

672

.

.

B

B

.

.

.

−0.83

*

.

.

−0.60

0.22

Val

673

.

.

B

B

.

.

.

−0.02

*

.

.

−0.30

0.39

Glu

674

.

.

B

B

.

.

.

−0.03

.

.

.

0.30

0.42

Ile

675

.

.

B

B

.

.

.

0.31

.

.

.

0.30

0.86

Glu

676

A

.

.

B

.

.

.

0.21

.

.

F

0.90

2.01

Asp

677

A

.

.

.

.

T

.

0.29

.

.

F

1.30

1.15

Thr

678

A

.

.

.

.

T

.

1.19

.

*

F

1.30

1.35

Glu

679

A

.

.

.

.

T

.

1.19

.

.

F

1.30

1.56

Gly

680

A

.

.

.

.

T

.

1.22

*

.

F

1.30

1.62

Leu

681

A

A

.

.

.

.

.

0.33

*

.

F

0.75

0.83

Lys

682

A

A

.

.

.

.

.

0.33

*

.

F

0.75

0.34

Glu

683

A

A

.

.

.

.

.

0.64

*

*

.

0.30

0.55

Val

684

A

A

.

.

.

.

.

0.76

*

*

.

0.75

1.11

Ile

685

A

A

.

.

.

.

.

0.86

*

*

.

0.75

1.09

Asn

686

A

.

.

.

.

T

.

1.67

*

*

.

1.00

0.98

Asp

687

A

.

.

.

.

T

.

1.02

*

*

.

1.15

2.21

Arg

688

A

.

.

.

.

T

.

0.21

*

.

.

1.15

3.12

Tyr

689

A

.

.

.

.

T

.

1.07

.

.

.

1.15

1.60

Asp

690

.

.

B

.

.

.

.

1.07

.

*

.

0.95

1.54

Met

691

.

.

B

B

.

.

.

0.77

.

.

.

−0.30

0.55

Leu

692

.

.

B

B

.

.

.

0.47

.

.

.

−0.60

0.47

Asn

693

.

.

B

B

.

.

.

−0.46

.

.

.

−0.30

0.38

Ile

694

.

.

B

B

.

.

.

−0.10

.

.

.

−0.60

0.32

Ser

695

.

.

B

B

.

.

.

−0.10

.

.

F

0.19

0.75

Ser

696

.

.

B

B

.

.

.

0.50

.

.

F

1.13

0.81

Leu

697

.

.

B

B

.

.

.

0.97

*

*

F

1.62

1.92

Arg

698

A

.

.

.

.

T

.

1.01

*

*

F

2.66

1.42

Gln

699

.

.

.

.

T

T

.

1.59

*

*

F

3.40

2.12

Asp

700

.

.

.

.

T

T

.

1.19

*

*

F

3.06

3.71

Gly

701

.

.

.

.

T

T

.

0.60

*

*

F

2.72

1.64

Lys

702

.

.

B

.

.

.

.

1.41

*

*

F

1.33

0.66

Thr

703

.

.

B

.

.

.

.

0.60

*

*

F

1.29

0.66

Phe

704

.

A

B

.

.

.

.

0.64

*

*

.

−0.30

0.58

Ile

705

.

A

B

.

.

.

.

0.69

*

*

.

0.30

0.58

Asp

706

A

A

.

.

.

.

.

0.79

*

*

.

0.64

0.80

Phe

707

A

A

.

.

.

.

.

0.74

*

*

.

0.53

1.46

Lys

708

A

A

.

.

.

.

.

1.06

*

.

F

1.62

3.34

Lys

709

.

A

.

.

T

.

.

1.80

*

.

F

2.66

3.34

Tyr

710

.

.

.

.

T

T

.

1.88

*

*

F

3.40

7.72

Asn

711

.

.

.

.

T

T

.

1.67

*

*

F

3.06

3.18

Asp

712

.

.

.

.

T

T

.

1.56

*

*

F

2.72

2.46

Lys

713

.

.

B

.

.

T

.

1.27

*

*

F

1.68

1.29

Leu

714

.

.

B

B

.

.

.

0.33

*

*

.

0.79

1.26

Pro

715

.

.

B

B

.

.

.

0.28

*

*

.

−0.30

0.53

Leu

716

.

.

B

B

.

.

.

0.28

*

*

.

−0.60

0.35

Tyr

717

.

.

B

B

.

.

.

0.07

*

.

.

−0.60

0.69

Ile

718

.

.

B

B

.

.

.

0.02

.

*

.

−0.60

0.69

Ser

719

.

.

B

B

.

.

.

0.59

.

*

.

−0.45

1.35

Asn

720

.

.

B

.

.

T

.

0.84

.

*

F

0.10

1.35

Pro

721

.

.

.

.

T

T

.

0.80

.

*

F

0.80

3.85

Asn

722

.

.

.

.

T

T

.

1.04

.

*

F

0.80

2.13

Tyr

723

.

.

.

.

T

T

.

1.08

.

*

F

0.80

2.13

Lys

724

.

.

B

B

.

.

.

1.13

.

*

.

−0.15

1.02

Val

725

.

.

B

B

.

.

.

0.54

.

*

.

−0.60

1.00

Asn

726

.

.

B

B

.

.

.

−0.10

.

*

.

−0.60

0.64

Val

727

.

.

B

B

.

.

.

−0.41

.

*

.

−0.60

0.24

Tyr

728

.

.

B

B

.

.

.

−0.12

.

*

.

−0.60

0.46

Ala

729

.

.

B

B

.

.

.

−0.17

.

*

.

−0.60

0.58

Val

730

A

.

.

B

.

.

.

0.69

.

*

.

−0.15

1.35

Thr

731

A

.

.

B

.

.

.

0.38

.

.

F

0.60

1.38

Lys

732

.

.

B

B

.

.

.

0.34

.

.

F

0.60

1.97

Glu

733

.

.

B

B

.

.

.

−0.30

*

.

F

0.60

1.86

Asn

734

.

.

B

B

.

.

.

0.29

*

.

F

0.45

0.91

Thr

735

.

.

B

B

.

.

.

0.93

.

.

F

0.45

0.73

Ile

736

.

.

B

B

.

.

.

0.94

.

.

.

−0.30

0.65

Ile

737

.

.

B

B

.

.

.

0.90

.

.

.

−0.26

0.54

Asn

738

.

.

B

.

.

T

.

0.90

*

.

F

0.93

0.65

Pro

739

.

.

.

.

.

T

C

0.56

*

.

F

2.22

1.49

Ser

740

.

.

.

.

.

T

C

0.87

*

.

F

2.56

2.11

Glu

741

.

.

.

.

T

T

.

1.44

*

.

F

3.40

2.19

Asn

742

.

.

.

.

T

T

.

2.03

.

*

F

3.06

2.04

Gly

743

.

.

.

.

T

T

.

1.72

.

.

F

2.72

2.04

Asp

744

.

.

.

.

T

T

.

1.93

.

.

F

2.38

1.70

Thr

745

.

.

.

.

.

T

C

1.89

*

.

F

1.54

1.70

Ser

746

.

.

.

.

.

T

C

1.00

*

.

F

1.20

1.70

Thr

747

A

.

.

.

.

T

.

1.04

*

.

F

0.85

0.71

Asn

748

A

.

.

.

.

T

.

1.43

*

.

F

0.85

0.99

Gly

749

A

.

.

.

.

T

.

0.54

*

.

F

1.30

1.48

Ile

750

A

.

.

B

.

.

.

0.04

*

.

F

0.45

0.72

Lys

751

.

.

B

B

.

.

.

−0.54

*

.

F

0.45

0.37

Lys

752

.

.

B

B

.

.

.

−0.93

*

.

F

−0.15

0.26

Ile

753

.

.

B

B

.

.

.

−1.23

*

.

.

−0.60

0.32

Leu

754

.

.

B

B

.

.

.

−0.84

*

.

.

−0.30

0.22

Ile

755

.

.

B

B

.

.

.

0.09

*

.

.

−0.30

0.22

Phe

756

.

.

B

B

.

.

.

−0.30

*

.

.

0.04

0.62

Ser

757

.

.

.

.

.

T

C

−0.59

*

.

F

1.73

0.74

Lys

758

.

.

.

.

T

T

.

0.30

*

.

F

1.82

1.65

Lys

759

.

.

.

.

.

T

C

0.22

*

.

F

2.86

3.30

Gly

760

.

.

.

.

T

T

.

0.77

*

.

F

3.40

1.73

Tyr

761

.

.

.

.

T

.

.

1.08

*

.

F

2.71

0.85

Glu

762

.

.

B

.

.

.

.

0.99

*

.

.

1.52

0.55

Ile

763

.

.

B

.

.

.

.

0.56

*

.

.

0.58

0.71

Gly

764

.

.

B

.

.

.

.

0.12

.

.

.

0.24

0.58

In another aspect, the invention provides an antibody that binds a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide described herein. The epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention.

As to the selection of peptides or polypeptides bearing an antigenic epitope (i.e., that contain a region of a protein molecule to which an antibody can bind), it is well known in that art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R. A. (1983) Antibodies that react with predetermined sites on proteins. Science 2/9:660-666. Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.

Antigenic epitope-bearing peptides and polypeptides are therefore useful to raise antibodies, including monoclonal antibodies, that bind to a PA polypeptide of the invention. See, for instance, Wilson et al., Cell 37:767-778 (1984) at 777. Antigenic epitope-bearing peptides and polypeptides preferably contain a sequence of at least seven, more preferably at least nine and most preferably between at least about 15 to about 30 amino acids contained within the amino acid sequence of SEQ ID NO:2.

Antibodies of the invention may bind one or more antigenic PA polypeptides or peptides including, but not limited to: a polypeptide comprising amino acid residues from about 39 to about 45 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 129 to about 134 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 151 to about 157 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 168 to about 172 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 189 to about 195 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 203 to about 213 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 225 to about 230 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 246 to about 253 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 259 to about 264 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 273 to about 280 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 302 to about 307 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 309 to about 314 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 319 to about 331 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 452 to about 457 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 480 to about 483 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 494 to about 498 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 523 to about 527 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 538 to about 544 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 564 to about 567 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 572 to about 575 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 587 to about 591 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 626 to about 631 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 650 to about 653 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 697 to about 701 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 708 to about 713 of SEQ ID NO:2; a polypeptide comprising amino acid residues from about 739 to about 745 of SEQ ID NO:2; and/or a polypeptide comprising amino acid residues from about 757 to about 762 of SEQ ID NO:2. In this context “about” includes the particularly recited range, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either terminus or at both termini. Epitope-bearing PA peptides and polypeptides may be produced by any conventional means. Houghten, R. A., “General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids,” Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985). This “Simultaneous Multiple Peptide Synthesis (SMPS)” process is further described in U.S. Pat. No. 4,631,211 to Houghten et al. (1986).

As one of skill in the art will appreciate, PA polypeptides and the epitope-bearing fragments thereof described herein can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EPA 394,827; Traunecker et al., Nature 331:84-86 (1988)). Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric PA protein or protein fragment alone (Fountoulakis et al., J Biochem 270:3958-3964 (1995)). Thus, antibodies of the invention may bind the PA moiety of fusion proteins that comprise all or a portion of a PA polypeptide.

Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or “muteins” including single or multiple amino acid substitutions, deletions, additions or fusion proteins. Such modified polypeptides can show, e.g., enhanced activity or increased stability. In addition, they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions. Antibodies of the present invention may also bind such modified PA polypeptides or PA polypeptide fragments or variants.

For instance, for many proteins, it is known in the art that one or more amino acids may be deleted from the N-terminus or C-terminus without substantial loss of biological function, or loss of the ability to be bound by a specific antibody. For instance, Ron et al., J. Biol. Chem., 268:2984-2988 (1993) reported modified KGF proteins that had heparin binding activity even if 3, 8, or 27 amino-terminal amino acid residues were missing.

However, even if deletion of one or more amino acids from the N-terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind EF or LF may still be retained. For example, the ability of shortened PA polypeptides to induce and/or bind to antibodies which recognize the complete or mature forms of the PA polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a PA polypeptide with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six PA amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the amino terminus of the PA amino acid sequence of SEQ ID NO:2 up to the serine residue at position number 463. In particular, the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues n1-764 of SEQ ID NO:2, where n1 is an integer from 31 to 759 corresponding to the position of the amino acid residue in SEQ ID NO:2.

More in particular, the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues of V-31 to G-764; K-32 to G-764; Q-33 to G-764; E-34 to G-764; N-35 to G-764; R-36 to G-764; L-37 to G-764; L-38 to G-764; N-39 to G-764; E-40 to G-764; S-41 to G-764; E-42 to G-764; S-43 to G-764; S-44 to G-764; S-45 to G-764; Q-46 to G-764; G-47 to G-764; L-48 to G-764; L-49 to G-764; G-50 to G-764; Y-51 to G-764; Y-52 to G-764; F-53 to G-764; S-54 to G-764; D-55 to G-764; L-56 to G-764; N-57 to G-764; F-58 to G-764; Q-59 to G-764; A-60 to G-764; P-61 to G-764; M-62 to G-764; V-63 to G-764; V-64 to G-764; T-65 to G-764; S-66 to G-764; S-67 to G-764; T-68 to G-764; T-69 to G-764; G-70 to G-764; D-71 to G-764; L-72 to G-764; S-73 to G-764; I-74 to G-764; P-75 to G-764; S-76 to G-764; S-77 to G-764; E-78 to G-764; L-79 to G-764; E-80 to G-764; N-81 to G-764; I-82 to G-764; P-83 to G-764; S-84 to G-764; E-85 to G-764; N-86 to G-764; Q-87 to G-764; Y-88 to G-764; F-89 to G-764; Q-90 to G-764; S-91 to G-764; A-92 to G-764; I-93 to G-764; W-94 to G-764; S-95 to G-764; G-96 to G-764; F-97 to G-764; I-98 to G-764; K-99 to G-764; V-100 to G-764; K-101 to G-764; K-102 to G-764; S-103 to G-764; D-104 to G-764; E-105 to G-764; Y-106 to G-764; T-107 to G-764; F-108 to G-764; A-109 to G-764; T-110 to G-764; S-111 to G-764; A-112 to G-764; D-113 to G-764; N-114 to G-764; H-115 to G-764; V-116 to G-764; I-117 to G-764; M-118 to G-764; W-119 to G-764; V-120 to G-764; D-121 to G-764; D-122 to G-764; Q-123 to G-764; E-124 to G-764; V-125 to G-764; I-126 to G-764; N-127 to G-764; K-128 to G-764; A-129 to G-764; S-130 to G-764; N-131 to G-764; S-132 to G-764; N-133 to G-764; K-134 to G-764; I-135 to G-764; R-136 to G-764; L-137 to G-764; E-138 to G-764; K-139 to G-764; G-140 to G-764; R-141 to G-764; L-142 to G-764; Y-143 to G-764; Q-144 to G-764; I-145 to G-764; K-146 to G-764; I-147 to G-764; Q-148 to G-764; Y-149 to G-764; Q-150 to G-764; R-151 to G-764; E-152 to G-764; N-153 to G-764; P-154 to G-764; T-155 to G-764; E-156 to G-764; K-157 to G-764; G-158 to G-764; L-159 to G-764; D-160 to G-764; F-161 to G-764; K-162 to G-764; L-163 to G-764; Y-164 to G-764; W-165 to G-764; T-166 to G-764; D-167 to G-764; S-168 to G-764; Q-169 to G-764; N-170 to G-764; K-171 to G-764; K-172 to G-764; E-173 to G-764; V-174 to G-764; I-175 to G-764; S-176 to G-764; S-177 to G-764; D-178 to G-764; N-179 to G-764; L-180 to G-764; Q-181 to G-764; L-182 to G-764; P-183 to G-764; E-184 to G-764; L-185 to G-764; K-186 to G-764; Q-187 to G-764; K-188 to G-764; S-189 to G-764; S-190 to G-764; N-191 to G-764; S-192 to G-764; R-193 to G-764; K-194 to G-764; K-195 to G-764; R-196 to G-764; S-197 to G-764; T-198 to G-764; S-199 to G-764; A-200 to G-764; G-201 to G-764; P-202 to G-764; T-203 to G-764; V-204 to G-764; P-205 to G-764; D-206 to G-764; R-207 to G-764; D-208 to G-764; N-209 to G-764; D-210 to G-764; G-211 to G-764; I-212 to G-764; P-213 to G-764; D-214 to G-764; S-215 to G-764; L-216 to G-764; E-217 to G-764; V-218 to G-764; E-219 to G-764; G-220 to G-764; Y-221 to G-764; T-222 to G-764; V-223 to G-764; D-224 to G-764; V-225 to G-764; K-226 to G-764; N-227 to G-764; K-228 to G-764; R-229 to G-764; T-230 to G-764; F-231 to G-764; L-232 to G-764; S-233 to G-764; P-234 to G-764; W-235 to G-764; I-236 to G-764; S-237 to G-764; N-238 to G-764; I-239 to G-764; H-240 to G-764; E-241 to G-764; K-242 to G-764; K-243 to G-764; G-244 to G-764; L-245 to G-764; T-246 to G-764; K-247 to G-764; Y-248 to G-764; K-249 to G-764; S-250 to G-764; S-251 to G-764; P-252 to G-764; E-253 to G-764; K-254 to G-764; W-255 to G-764; S-256 to G-764; T-257 to G-764; A-258 to G-764; S-259 to G-764; D-260 to G-764; P-261 to G-764; Y-262 to G-764; S-263 to G-764; D-264 to G-764; F-265 to G-764; E-266 to G-764; K-267 to G-764; V-268 to G-764; T-269 to G-764; G-270 to G-764; R-271 to G-764; I-272 to G-764; D-273 to G-764; K-274 to G-764; N-275 to G-764; V-276 to G-764; S-277 to G-764; P-278 to G-764; E-279 to G-764; A-280 to G-764; R-281 to G-764; H-282 to G-764; P-283 to G-764; L-284 to G-764; V-285 to G-764; A-286 to G-764; A-287 to G-764; Y-288 to G-764; P-289 to G-764; I-290 to G-764; V-291 to G-764; H-292 to G-764; V-293 to G-764; D-294 to G-764; M-295 to G-764; E-296 to G-764; N-297 to G-764; I-298 to G-764; I-299 to G-764; L-300 to G-764; S-301 to G-764; K-302 to G-764; N-303 to G-764; E-304 to G-764; D-305 to G-764; Q-306 to G-764; S-307 to G-764; T-308 to G-764; Q-309 to G-764; N-310 to G-764; T-311 to G-764; D-312 to G-764; S-313 to G-764; Q-314 to G-764; T-315 to G-764; R-316 to G-764; T-317 to G-764; I-318 to G-764; S-319 to G-764; K-320 to G-764; N-321 to G-764; T-322 to G-764; S-323 to G-764; T-324 to G-764; S-325 to G-764; R-326 to G-764; T-327 to G-764; H-328 to G-764; T-329 to G-764; S-330 to G-764; E-331 to G-764; V-332 to G-764; H-333 to G-764; G-334 to G-764; N-335 to G-764; A-336 to G-764; E-337 to G-764; V-338 to G-764; H-339 to G-764; A-340 to G-764; S-341 to G-764; F-342 to G-764; F-343 to G-764; D-344 to G-764; I-345 to G-764; G-346 to G-764; G-347 to G-764; S-348 to G-764; V-349 to G-764; S-350 to G-764; A-351 to G-764; G-352 to G-764; F-353 to G-764; S-354 to G-764; N-355 to G-764; S-356 to G-764; N-357 to G-764; S-358 to G-764; S-359 to G-764; T-360 to G-764; V-361 to G-764; A-362 to G-764; I-363 to G-764; D-364 to G-764; H-365 to G-764; S-366 to G-764; L-367 to G-764; S-368 to G-764; L-369 to G-764; A-370 to G-764; G-371 to G-764; E-372 to G-764; R-373 to G-764; T-374 to G-764; W-375 to G-764; A-376 to G-764; E-377 to G-764; T-378 to G-764; M-379 to G-764; G-380 to G-764; L-381 to G-764; N-382 to G-764; T-383 to G-764; A-384 to G-764; D-385 to G-764; T-386 to G-764; A-387 to G-764; R-388 to G-764; L-389 to G-764; N-390 to G-764; A-391 to G-764; N-392 to G-764; I-393 to G-764; R-394 to G-764; Y-395 to G-764; V-396 to G-764; N-397 to G-764; T-398 to G-764; G-399 to G-764; T-400 to G-764; A-401 to G-764; P-402 to G-764; I-403 to G-764; Y-404 to G-764; N-405 to G-764; V-406 to G-764; L-407 to G-764; P-408 to G-764; T-409 to G-764; T-410 to G-764; S-411 to G-764; L-412 to G-764; V-413 to G-764; L-414 to G-764; G-415 to G-764; K-416 to G-764; N-417 to G-764; Q-418 to G-764; T-419 to G-764; L-420 to G-764; A-421 to G-764; T-422 to G-764; I-423 to G-764; K-424 to G-764; A-425 to G-764; K-426 to G-764; E-427 to G-764; N-428 to G-764; Q-429 to G-764; L-430 to G-764; S-431 to G-764; Q-432 to G-764; I-433 to G-764; L-434 to G-764; A-435 to G-764; P-436 to G-764; N-437 to G-764; N-438 to G-764; Y-439 to G-764; Y-440 to G-764; P-441 to G-764; S-442 to G-764; K-443 to G-764; N-444 to G-764; L-445 to G-764; A-446 to G-764; P-447 to G-764; I-448 to G-764; A-449 to G-764; L-450 to G-764; N-451 to G-764; A-452 to G-764; Q-453 to G-764; D-454 to G-764; D-455 to G-764; F-456 to G-764; S-457 to G-764; S-458 to G-764; T-459 to G-764; P-460 to G-764; I-461 to G-764; T-462 to G-764; M-463 to G-764; N-464 to G-764; Y-465 to G-764; N-466 to G-764; Q-467 to G-764; F-468 to G-764; L-469 to G-764; E-470 to G-764; L-471 to G-764; E-472 to G-764; K-473 to G-764; T-474 to G-764; K-475 to G-764; Q-476 to G-764; L-477 to G-764; R-478 to G-764; L-479 to G-764; D-480 to G-764; T-481 to G-764; D-482 to G-764; Q-483 to G-764; V-484 to G-764; Y-485 to G-764; G-486 to G-764; N-487 to G-764; I-488 to G-764; A-489 to G-764; T-490 to G-764; Y-491 to G-764; N-492 to G-764; F-493 to G-764; E-494 to G-764; N-495 to G-764; G-496 to G-764; R-497 to G-764; V-498 to G-764; R-499 to G-764; V-500 to G-764; D-501 to G-764; T-502 to G-764; G-503 to G-764; S-504 to G-764; N-505 to G-764; W-506 to G-764; S-507 to G-764; E-508 to G-764; V-509 to G-764; L-510 to G-764; P-511 to G-764; Q-512 to G-764; I-513 to G-764; Q-514 to G-764; E-515 to G-764; T-516 to G-764; T-517 to G-764; A-518 to G-764; R-519 to G-764; I-520 to G-764; I-521 to G-764; F-522 to G-764; N-523 to G-764; G-524 to G-764; K-525 to G-764; D-526 to G-764; L-527 to G-764; N-528 to G-764; L-529 to G-764; V-530 to G-764; E-531 to G-764; R-532 to G-764; R-533 to G-764; I-534 to G-764; A-535 to G-764; A-536 to G-764; V-537 to G-764; N-538 to G-764; P-539 to G-764; S-540 to G-764; D-541 to G-764; P-542 to G-764; L-543 to G-764; E-544 to G-764; T-545 to G-764; T-546 to G-764; K-547 to G-764; P-548 to G-764; D-549 to G-764; M-550 to G-764; T-551 to G-764; L-552 to G-764; K-553 to G-764; E-554 to G-764; A-555 to G-764; L-556 to G-764; K-557 to G-764; I-558 to G-764; A-559 to G-764; F-560 to G-764; G-561 to G-764; F-562 to G-764; N-563 to G-764; E-564 to G-764; P-565 to G-764; N-566 to G-764; G-567 to G-764; N-568 to G-764; L-569 to G-764; Q-570 to G-764; Y-571 to G-764; Q-572 to G-764; G-573 to G-764; K-574 to G-764; D-575 to G-764; I-576 to G-764; I-577 to G-764; E-578 to G-764; F-579 to G-764; D-580 to G-764; F-581 to G-764; N-582 to G-764; F-583 to G-764; D-584 to G-764; Q-585 to G-764; Q-586 to G-764; T-587 to G-764; S-588 to G-764; Q-589 to G-764; N-590 to G-764; I-591 to G-764; K-592 to G-764; N-593 to G-764; Q-594 to G-764; L-595 to G-764; A-596 to G-764; E-597 to G-764; L-598 to G-764; N-599 to G-764; A-600 to G-764; T-601 to G-764; N-602 to G-764; I-603 to G-764; Y-604 to G-764; T-605 to G-764; V-606 to G-764; L-607 to G-764; D-608 to G-764; K-609 to G-764; I-610 to G-764; K-611 to G-764; L-612 to G-764; N-613 to G-764; A-614 to G-764; K-615 to G-764; M-616 to G-764; N-617 to G-764; I-618 to G-764; L-619 to G-764; I-620 to G-764; R-621 to G-764; D-622 to G-764; K-623 to G-764; R-624 to G-764; F-625 to G-764; H-626 to G-764; Y-627 to G-764; D-628 to G-764; R-629 to G-764; N-630 to G-764; N-631 to G-764; I-632 to G-764; A-633 to G-764; V-634 to G-764; G-635 to G-764; A-636 to G-764; D-637 to G-764; E-638 to G-764; S-639 to G-764; V-640 to G-764; V-641 to G-764; K-642 to G-764; E-643 to G-764; A-644 to G-764; H-645 to G-764; R-646 to G-764; E-647 to G-764; V-648 to G-764; I-649 to G-764; N-650 to G-764; S-651 to G-764; S-652 to G-764; T-653 to G-764; E-654 to G-764; G-655 to G-764; L-656 to G-764; L-657 to G-764; L-658 to G-764; N-659 to G-764; I-660 to G-764; D-661 to G-764; K-662 to G-764; D-663 to G-764; I-664 to G-764; R-665 to G-764; K-666 to G-764; I-667 to G-764; L-668 to G-764; S-669 to G-764; G-670 to G-764; Y-671 to G-764; I-672 to G-764; V-673 to G-764; E-674 to G-764; I-675 to G-764; E-676 to G-764; D-677 to G-764; T-678 to G-764; E-679 to G-764; G-680 to G-764; L-681 to G-764; K-682 to G-764; E-683 to G-764; V-684 to G-764; I-685 to G-764; N-686 to G-764; D-687 to G-764; R-688 to G-764; Y-689 to G-764; D-690 to G-764; M-691 to G-764; L-692 to G-764; N-693 to G-764; I-694 to G-764; S-695 to G-764; S-696 to G-764; L-697 to G-764; R-698 to G-764; Q-699 to G-764; D-700 to G-764; G-701 to G-764; K-702 to G-764; T-703 to G-764; F-704 to G-764; I-705 to G-764; D-706 to G-764; F-707 to G-764; K-708 to G-764; K-709 to G-764; Y-710 to G-764; N-711 to G-764; D-712 to G-764; K-713 to G-764; L-714 to G-764; P-715 to G-764; L-716 to G-764; Y-717 to G-764; I-718 to G-764; S-719 to G-764; N-720 to G-764; P-721 to G-764; N-722 to G-764; Y-723 to G-764; K-724 to G-764; V-725 to G-764; N-726 to G-764; V-727 to G-764; Y-728 to G-764; A-729 to G-764; V-730 to G-764; T-731 to G-764; K-732 to G-764; E-733 to G-764; N-734 to G-764; T-735 to G-764; I-736 to G-764; I-737 to G-764; N-738 to G-764; P-739 to G-764; S-740 to G-764; E-741 to G-764; N-742 to G-764; G-743 to G-764; D-744 to G-764; T-745 to G-764; S-746 to G-764; T-747 to G-764; N-748 to G-764; G-749 to G-764; I-750 to G-764; K-751 to G-764; K-752 to G-764; I-753 to G-764; L-754 to G-764; I-755 to G-764; F-756 to G-764; S-757 to G-764; K-758 to G-764; and/or K-759 to G-764; of the amino acid sequence of SEQ ID NO:2.

As mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind EF or LF) may still be retained. For example, the ability of the shortened PA polypeptide to induce and/or bind to antibodies which recognize the complete or mature forms of the PA polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a PA polypeptide with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six PA amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides antibodies that bind polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the PA polypeptide sequence of SEQ ID NO:2 up to the arginine residue at position number 36. In particular, the present invention provides antibodies that bind polypeptides comprising the amino acid sequence of residues 30-m1 of SEQ ID NO:2, where m1 is an integer from 36 to 763 corresponding to the position of the amino acid residue in SEQ ID NO:2.

More in particular, the invention provides antibodies that bind polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues E-30 to I-763; E-30 to E-762; E-30 to Y-761; E-30 to G-760; E-30 to K-759; E-30 to K-758; E-30 to S-757; E-30 to F-756; E-30 to I-755; E-30 to L-754; E-30 to I-753; E-30 to K-752; E-30 to K-751; E-30 to I-750; E-30 to G-749; E-30 to N-748; E-30 to T-747; E-30 to S-746; E-30 to T-745; E-30 to D-744; E-30 to G-743; E-30 to N-742; E-30 to E-741; E-30 to S-740; E-30 to P-739; E-30 to N-738; E-30 to I-737; E-30 to I-736; E-30 to T-735; E-30 to N-734; E-30 to E-733; E-30 to K-732; E-30 to T-731; E-30 to V-730; E-30 to A-729; E-30 to Y-728; E-30 to V-727; E-30 to N-726; E-30 to V-725; E-30 to K-724; E-30 to Y-723; E-30 to N-722; E-30 to P-721; E-30 to N-720; E-30 to S-719; E-30 to I-718; E-30 to Y-717; E-30 to L-716; E-30 to P-715; E-30 to L-714; E-30 to K-713; E-30 to D-712; E-30 to N-711; E-30 to Y-710; E-30 to K-709; E-30 to K-708; E-30 to F-707; E-30 to D-706; E-30 to I-705; E-30 to F-704; E-30 to T-703; E-30 to K-702; E-30 to G-701; E-30 to D-700; E-30 to Q-699; E-30 to R-698; E-30 to L-697; E-30 to S-696; E-30 to S-695; E-30 to I-694; E-30 to N-693; E-30 to L-692; E-30 to M-691; E-30 to D-690; E-30 to Y-689; E-30 to R-688; E-30 to D-687; E-30 to N-686; E-30 to I-685; E-30 to V-684; E-30 to E-683; E-30 to K-682; E-30 to L-681; E-30 to G-680; E-30 to E-679; E-30 to T-678; E-30 to D-677; E-30 to E-676; E-30 to I-675; E-30 to E-674; E-30 to V-673; E-30 to I-672; E-30 to Y-671; E-30 to G-670; E-30 to S-669; E-30 to L-668; E-30 to I-667; E-30 to K-666; E-30 to R-665; E-30 to I-664; E-30 to D-663; E-30 to K-662; E-30 to D-661; E-30 to I-660; E-30 to N-659; E-30 to L-658; E-30 to L-657; E-30 to L-656; E-30 to G-655; E-30 to E-654; E-30 to T-653; E-30 to S-652; E-30 to S-651; E-30 to N-650; E-30 to I-649; E-30 to V-648; E-30 to E-647; E-30 to R-646; E-30 to H-645; E-30 to A-644; E-30 to E-643; E-30 to K-642; E-30 to V-641; E-30 to V-640; E-30 to S-639; E-30 to E-638; E-30 to D-637; E-30 to A-636; E-30 to G-635; E-30 to V-634; E-30 to A-633; E-30 to I-632; E-30 to N-631; E-30 to N-630; E-30 to R-629; E-30 to D-628; E-30 to Y-627; E-30 to H-626; E-30 to F-625; E-30 to R-624; E-30 to K-623; E-30 to D-622; E-30 to R-621; E-30 to I-620; E-30 to L-619; E-30 to I-618; E-30 to N-617; E-30 to M-616; E-30 to K-615; E-30 to A-614; E-30 to N-613; E-30 to L-612; E-30 to K-611; E-30 to I-610; E-30 to K-609; E-30 to D-608; E-30 to L-607; E-30 to V-606; E-30 to T-605; E-30 to Y-604; E-30 to I-603; E-30 to N-602; E-30 to T-601; E-30 to A-600; E-30 to N-599; E-30 to L-598; E-30 to E-597; E-30 to A-596; E-30 to L-595; E-30 to Q-594; E-30 to N-593; E-30 to K-592; E-30 to I-591; E-30 to N-590; E-30 to Q-589; E-30 to S-588; E-30 to T-587; E-30 to Q-586; E-30 to Q-585; E-30 to D-584; E-30 to F-583; E-30 to N-582; E-30 to F-581; E-30 to D-580; E-30 to F-579; E-30 to E-578; E-30 to T-577; E-30 to I-576; E-30 to D-575; E-30 to K-574; E-30 to G-573; E-30 to Q-572; E-30 to Y-571; E-30 to Q-570; E-30 to L-569; E-30 to N-568; E-30 to G-567; E-30 to N-566; E-30 to P-565; E-30 to E-564; E-30 to N-563; E-30 to F-562; E-30 to G-561; E-30 to F-560; E-30 to A-559; E-30 to I-558; E-30 to K-557; E-30 to L-556; E-30 to A-555; E-30 to E-554; E-30 to K-553; E-30 to L-552; E-30 to T-551; E-30 to M-550; E-30 to D-549; E-30 to P-548; E-30 to K-547; E-30 to T-546; E-30 to T-545; E-30 to E-544; E-30 to L-543; E-30 to P-542; E-30 to D-541; E-30 to S-540; E-30 to P-539; E-30 to N-538; E-30 to V-537; E-30 to A-536; E-30 to A-535; E-30 to I-534; E-30 to R-533; E-30 to R-532; E-30 to E-531; E-30 to V-530; E-30 to L-529; E-30 to N-528; E-30 to L-527; E-30 to D-526; E-30 to K-525; E-30 to G-524; E-30 to N-523; E-30 to F-522; E-30 to I-521; E-30 to I-520; E-30 to R-519; E-30 to A-518; E-30 to T-517; E-30 to T-516; E-30 to E-515; E-30 to Q-514; E-30 to I-513; E-30 to Q-512; E-30 to P-511; E-30 to L-510; E-30 to V-509; E-30 to E-508; E-30 to S-507; E-30 to W-506; E-30 to N-505; E-30 to S-504; E-30 to G-503; E-30 to T-502; E-30 to D-501; E-30 to V-500; E-30 to R-499; E-30 to V-498; E-30 to R-497; E-30 to G-496; E-30 to N-495; E-30 to E-494; E-30 to F-493; E-30 to N-492; E-30 to Y-491; E-30 to T-490; E-30 to A-489; E-30 to I-488; E-30 to N-487; E-30 to G-486; E-30 to Y-485; E-30 to V-484; E-30 to Q-483; E-30 to D-482; E-30 to T-481; E-30 to D-480; E-30 to L-479; E-30 to R-478; E-30 to L-477; E-30 to Q-476; E-30 to K-475; E-30 to T-474; E-30 to K-473; E-30 to E-472; E-30 to L-471; E-30 to E-470; E-30 to L-469; E-30 to F-468; E-30 to Q-467; E-30 to N-466; E-30 to Y-465; E-30 to N-464; E-30 to M-463; E-30 to T-462; E-30 to I-461; E-30 to P-460; E-30 to T-459; E-30 to S-458; E-30 to S-457; E-30 to F-456; E-30 to D-455; E-30 to D-454; E-30 to Q-453; E-30 to A-452; E-30 to N-451; E-30 to L-450; E-30 to A-449; E-30 to I-448; E-30 to P-447; E-30 to A-446; E-30 to L-445; E-30 to N-444; E-30 to K-443; E-30 to S-442; E-30 to P-441; E-30 to Y-440; E-30 to Y-439; E-30 to N-438; E-30 to N-437; E-30 to P-436; E-30 to A-435; E-30 to L-434; E-30 to I-433; E-30 to Q-432; E-30 to S-431; E-30 to L-430; E-30 to Q-429; E-30 to N-428; E-30 to E-427; E-30 to K-426; E-30 to A-425; E-30 to K-424; E-30 to I-423; E-30 to T-422; E-30 to A-421; E-30 to L-420; E-30 to T-419; E-30 to Q-418; E-30 to N-417; E-30 to K-416; E-30 to G-415; E-30 to L-414; E-30 to V-413; E-30 to L-412; E-30 to S-411; E-30 to T-410; E-30 to T-409; E-30 to P-408; E-30 to L-407; E-30 to V-406; E-30 to N-405; E-30 to Y-404; E-30 to I-403; E-30 to P-402; E-30 to A-401; E-30 to T-400; E-30 to G-399; E-30 to T-398; E-30 to N-397; E-30 to V-396; E-30 to Y-395; E-30 to R-394; E-30 to I-393; E-30 to N-392; E-30 to A-391; E-30 to N-390; E-30 to L-389; E-30 to R-388; E-30 to A-387; E-30 to T-386; E-30 to D-385; E-30 to A-384; E-30 to T-383; E-30 to N-382; E-30 to L-381; E-30 to G-380; E-30 to M-379; E-30 to T-378; E-30 to E-377; E-30 to A-376; E-30 to W-375; E-30 to T-374; E-30 to R-373; E-30 to E-372; E-30 to G-371; E-30 to A-370; E-30 to L-369; E-30 to S-368; E-30 to L-367; E-30 to S-366; E-30 to H-365; E-30 to D-364; E-30 to I-363; E-30 to A-362; E-30 to V-361; E-30 to T-360; E-30 to S-359; E-30 to S-358; E-30 to N-357; E-30 to S-356; E-30 to N-355; E-30 to S-354; E-30 to F-353; E-30 to G-352; E-30 to A-351; E-30 to S-350; E-30 to V-349; E-30 to S-348; E-30 to G-347; E-30 to G-346; E-30 to I-345; E-30 to D-344; E-30 to F-343; E-30 to F-342; E-30 to S-341; E-30 to A-340; E-30 to H-339; E-30 to V-338; E-30 to E-337; E-30 to A-336; E-30 to N-335; E-30 to G-334; E-30 to H-333; E-30 to V-332; E-30 to E-331; E-30 to S-330; E-30 to T-329; E-30 to H-328; E-30 to T-327; E-30 to R-326; E-30 to S-325; E-30 to T-324; E-30 to S-323; E-30 to T-322; E-30 to N-321; E-30 to K-320; E-30 to S-319; E-30 to I-318; E-30 to T-317; E-30 to R-316; E-30 to T-315; E-30 to Q-314; E-30 to S-313; E-30 to D-312; E-30 to T-311; E-30 to N-310; E-30 to Q-309; E-30 to T-308; E-30 to S-307; E-30 to Q-306; E-30 to D-305; E-30 to E-304; E-30 to N-303; E-30 to K-302; E-30 to S-301; E-30 to L-300; E-30 to I-299; E-30 to I-298; E-30 to N-297; E-30 to E-296; E-30 to M-295; E-30 to D-294; E-30 to V-293; E-30 to H-292; E-30 to V-291; E-30 to I-290; E-30 to P-289; E-30 to Y-288; E-30 to A-287; E-30 to A-286; E-30 to V-285; E-30 to L-284; E-30 to P-283; E-30 to H-282; E-30 to R-281; E-30 to A-280; E-30 to E-279; E-30 to P-278; E-30 to S-277; E-30 to V-276; E-30 to N-275; E-30 to K-274; E-30 to D-273; E-30 to I-272; E-30 to R-271; E-30 to G-270; E-30 to T-269; E-30 to V-268; E-30 to K-267; E-30 to E-266; E-30 to F-265; E-30 to D-264; E-30 to S-263; E-30 to Y-262; E-30 to P-261; E-30 to D-260; E-30 to S-259; E-30 to A-258; E-30 to T-257; E-30 to S-256; E-30 to W-255; E-30 to K-254; E-30 to E-253; E-30 to P-252; E-30 to S-251; E-30 to S-250; E-30 to K-249; E-30 to Y-248; E-30 to K-247; E-30 to T-246; E-30 to L-245; E-30 to G-244; E-30 to K-243; E-30 to K-242; E-30 to E-241; E-30 to H-240; E-30 to I-239; E-30 to N-238; E-30 to S-237; E-30 to I-236; E-30 to W-235; E-30 to P-234; E-30 to S-233; E-30 to L-232; E-30 to F-231; E-30 to T-230; E-30 to R-229; E-30 to K-228; E-30 to N-227; E-30 to K-226; E-30 to V-225; E-30 to D-224; E-30 to V-223; E-30 to T-222; E-30 to Y-221; E-30 to G-220; E-30 to E-219; E-30 to V-218; E-30 to E-217; E-30 to L-216; E-30 to S-215; E-30 to D-214; E-30 to P-213; E-30 to I-212; E-30 to G-211; E-30 to D-210; E-30 to N-209; E-30 to D-208; E-30 to R-207; E-30 to D-206; E-30 to P-205; E-30 to V-204; E-30 to T-203; E-30 to P-202; E-30 to G-201; E-30 to A-200; E-30 to S-199; E-30 to T-198; E-30 to S-197; E-30 to R-196; E-30 to K-195; E-30 to K-194; E-30 to R-193; E-30 to S-192; E-30 to N-191; E-30 to S-190; E-30 to S-189; E-30 to K-188; E-30 to Q-187; E-30 to K-186; E-30 to L-185; E-30 to E-184; E-30 to P-183; E-30 to L-182; E-30 to Q-181; E-30 to L-180; E-30 to N-179; E-30 to D-178; E-30 to S-177; E-30 to S-176; E-30 to I-175; E-30 to V-174; E-30 to E-173; E-30 to K-172; E-30 to K-171; E-30 to N-170; E-30 to Q-169; E-30 to S-168; E-30 to D-167; E-30 to T-166; E-30 to W-165; E-30 to Y-164; E-30 to L-163; E-30 to K-162; E-30 to F-161; E-30 to D-160; E-30 to L-159; E-30 to G-158; E-30 to K-157; E-30 to E-156; E-30 to T-155; E-30 to P-154; E-30 to N-153; E-30 to E-152; E-30 to R-151; E-30 to Q-150; E-30 to Y-149; E-30 to Q-148; E-30 to I-147; E-30 to K-146; E-30 to I-145; E-30 to Q-144; E-30 to Y-143; E-30 to L-142; E-30 to R-141; E-30 to G-140; E-30 to K-139; E-30 to E-138; E-30 to L-137; E-30 to R-136; E-30 to I-135; E-30 to K-134; E-30 to N-133; E-30 to S-132; E-30 to N-131; E-30 to S-130; E-30 to A-129; E-30 to K-128; E-30 to N-127; E-30 to I-126; E-30 to V-125; E-30 to E-124; E-30 to Q-123; E-30 to D-122; E-30 to D-121; E-30 to V-120; E-30 to W-119; E-30 to M-118; E-30 to T-117; E-30 to V-116; E-30 to H-115; E-30 to N-114; E-30 to D-113; E-30 to A-112; E-30 to S-111; E-30 to T-110; E-30 to A-109; E-30 to F-108; E-30 to T-107; E-30 to Y-106; E-30 to E-105; E-30 to D-104; E-30 to S-103; E-30 to K-102; E-30 to K-101; E-30 to V-100; E-30 to K-99; E-30 to I-98; E-30 to F-97; E-30 to G-96; E-30 to S-95; E-30 to W-94; E-30 to I-93; E-30 to A-92; E-30 to S-91; E-30 to Q-90; E-30 to F-89; E-30 to Y-88; E-30 to Q-87; E-30 to N-86; E-30 to E-85; E-30 to S-84; E-30 to P-83; E-30 to I-82; E-30 to N-81; E-30 to E-80; E-30 to L-79; E-30 to E-78; E-30 to S-77; E-30 to S-76; E-30 to P-75; E-30 to I-74; E-30 to S-73; E-30 to L-72; E-30 to D-71; E-30 to G-70; E-30 to T-69; E-30 to T-68; E-30 to S-67; E-30 to S-66; E-30 to T-65; E-30 to V-64; E-30 to V-63; E-30 to M-62; E-30 to P-61; E-30 to A-60; E-30 to Q-59; E-30 to F-58; E-30 to N-57; E-30 to L-56; E-30 to D-55; E-30 to S-54; E-30 to F-53; E-30 to Y-52; E-30 to Y-51; E-30 to G-50; E-30 to L-49; E-30 to L-48; E-30 to G-47; E-30 to Q-46; E-30 to S-45; E-30 to S-44; E-30 to S-43; E-30 to E-42; E-30 to S-41; E-30 to E-40; E-30 to N-39; E-30 to L-38; E-30 to L-37; and/or E-30 to R-36 of the amino acid sequence of SEQ ID NO:2.

The invention also provides antibodies that bind polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a PA polypeptide, which may be described generally as having residues n1-m1 of SEQ ID NO:2, where n1 and m1 are integers as described above.

It will be recognized in the art that some amino acid sequence of PA can be varied without significant effect of the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine activity. Such areas will usually comprise residues which make up the ligand binding site or the death domain, or which form tertiary structures which affect these domains.

Thus, the invention further includes antibodies that bind variations of the PA protein which show substantial PA protein activity or which include regions of PA such as the protein fragments discussed below. Such mutants include deletions, insertions, inversions, repeats, and type substitution. Guidance concerning which amino acid changes are likely to be phenotypically silent can be found in Bowie, J. U. et al., Science 247:1306-1310 (1990).

Thus, antibodies of the present invention may bind a fragment, derivative, or analog of the polypeptide of SEQ ID NO:2. Such fragments, variants or derivatives may be (i) one in which at least one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue(s), and more preferably at least one but less than ten conserved amino acid residues) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

Of particular interest are substitutions of charged amino acids with another charged amino acid and with neutral or negatively charged amino acids. The latter results in proteins with reduced positive charge to improve the characteristics of the PA protein. The prevention of aggregation is highly desirable. Aggregation of proteins not only results in a loss of activity but can also be problematic when preparing pharmaceutical formulations, because they can be immunogenic. (Pinckard et al., Clin Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993)).

The replacement of amino acids can also change the selectivity of binding to cell surface receptors. Ostade et al., Nature 361:266-268 (1993) describes certain mutations resulting in selective binding of TNF-alpha to only one of the two known types of TNF receptors. Thus, the antibodies of the present invention may bind a PA protein that contains one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation.

As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table 3).

Amino acids in the PA protein that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as receptor binding or protein multimerization, pore formation, and toxin translocation. Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)). In preferred embodiments, antibodies of the present invention bind regions of PA that are essential for PA function. In other preferred embodiments, antibodies of the present invention bind regions of PA that are essential for PA function and inhibit or abolish PA function.

Additionally, protein engineering, may be employed to improve or alter the characteristics of PA polypeptides. Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or muteins including single or multiple amino acid substitutions, deletions, additions or fusion proteins. Such modified polypeptides can show, e.g., enhanced activity or increased stability. In addition, they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions. Antibodies of the present invention may bind such modified PA polypeptides.

Thus, the invention also encompasses antibodies that bind PA derivatives and analogs that have one or more amino acid residues deleted, added, and/or substituted. For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges; N-linked glycosylation sites can be altered or eliminated to achieve, for example, expression of a homogeneous product that is more easily recovered and purified from yeast hosts which are known to hyperglycosylate N-linked sites. To this end, a variety of amino acid substitutions at one or both of the first or third amino acid positions on any one or more of the glycosylation recognition sequences in the PA polypeptides and/or an amino acid deletion at the second position of any one or more such recognition sequences will prevent glycosylation of the PA at the modified tripeptide sequence (see, e.g., Miyajimo et al., EMBO J 5(6):1193-1197). Additionally, one or more of the amino acid residues of PA polypeptides (e.g., arginine and lysine residues) may be deleted or substituted with another residue to eliminate undesired processing by proteases such as, for example, furins or kexins.

The antibodies of the present invention also include antibodies that bind a polypeptide comprising, or alternatively, consisting of a polypeptide comprising, or alternatively, consisting of the polypeptide of SEQ ID NO:2 including the leader; a polypeptide comprising, or alternatively, consisting of the polypeptide of SEQ ID NO:2 minus the amino terminal methionine; a polypeptide comprising, or alternatively, consisting of the polypeptide of SEQ ID NO:2 minus the leader; a polypeptide comprising, or alternatively, consisting of the PA domain I; a polypeptide comprising, or alternatively, consisting of the PA domain II; a polypeptide comprising, or alternatively, consisting of the PA domain III; a polypeptide comprising, or alternatively, consisting of the PA domain IV; a polypeptide comprising, or alternatively, consisting of the PA20 fragment; a polypeptide comprising, or alternatively, consisting of the PA63 fragment; as well as polypeptides which are at least 80% identical, more preferably at least 90% or 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to the polypeptides described above (the polypeptide and polypeptide fragments of SEQ ID NO:2), and portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids.

By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a reference amino acid sequence of a PA polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the PA polypeptide. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in SEQ ID NO:2 can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.

In a specific embodiment, the identity between a reference (query) sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter. According to this embodiment, if the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction is made to the results to take into consideration the fact that the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. A determination of whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of this embodiment. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence. For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are made for the purposes of this embodiment.

The present application is also directed to antibodies that bind proteins containing polypeptides at least 90%, 95%, 96%, 97%, 98% or 99% identical to the PA polypeptide sequence set forth herein as n1-m1. In preferred embodiments, the present invention encompasses antibodies that bind proteins containing polypeptides at least 90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the specific PA N- and C-terminal deletions recited herein.

In certain preferred embodiments, antibodies of the invention bind PA fusion proteins as described above wherein the PA portion of the fusion protein are those described as n1-m1 herein.

Antibodies of the Invention May Bind Modified PA Polypeptides

It is specifically contemplated that antibodies of the present invention may bind modified forms of PA proteins SEQ ID NO:2). In specific embodiments, antibodies of the present invention bind PA polypeptides (such as those described above) including, but not limited to naturally purified PA polypeptides, PA polypeptides produced by chemical synthetic procedures, and PA polypeptides produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells using, for example, the recombinant compositions and methods described above. Depending upon the host employed in a recombinant production procedure, the polypeptides may be glycosylated or non-glycosylated. In addition, PA polypeptides may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.

The invention additionally encompasses antibodies that bind PA polypeptides that are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to, specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin; etc.

Additional post-translational modifications to PA polypeptides for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression. The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.

Also provided by the invention are antibodies that bind chemically modified derivatives of PA polypeptides which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kilodalton and about 100 kilodalton (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog). For example, the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kilodalton.

The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues, glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to a proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.

One may specifically desire proteins chemically modified at the N-terminus. Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.

As indicated above, pegylation of the proteins of the invention may be accomplished by any number of means. For example, polyethylene glycol may be attached to the protein either directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998); U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by reference.

One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (ClSO2CH2CF3). Upon reaction of protein with tresylated MPEG, polyethylene glycol is directly attached to amine groups of the protein. Thus, the invention includes protein-polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.

Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, U.S. Pat. No. 5,612,460, the entire disclosure of which is incorporated herein by reference, discloses urethane linkers for connecting polyethylene glycol to proteins. Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. A number additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in WO 98/32466, the entire disclosure of which is incorporated herein by reference. Pegylated protein products produced using the reaction chemistries set out herein are included within the scope of the invention.

The number of polyethylene glycol moieties attached to each PA polypeptide (i.e., the degree of substitution) may also vary. For example, the pegylated proteins of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

As mentioned the antibodies of the present invention may bind PA polypeptides that are modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given PA polypeptide. PA polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic PA polypeptides may result from posttranslational natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation. GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992)).

Anti-PA Antibodies

In one embodiment, the invention provides antibodies (e.g., antibodies comprising two heavy chains and two light chains linked together by disulfide bridges) that specifically bind PA (SEQ ID NO:2) or fragments or variants thereof, wherein the amino acid sequence of the heavy chain and the amino acid sequence of the light chain are the same as the amino acid sequence of a heavy chain and a light chain of one or more scFvs or cell lines referred to in Table 1. In another embodiment, the invention provides antibodies (each consisting of two heavy chains and two light chains linked together by disulfide bridges to form an antibody) that specifically bind PA or fragments or variants thereof, wherein the amino acid sequence of the heavy chain or the amino acid sequence of the light chain are the same as the amino acid sequence of a heavy chain or a light chain of one or more scFvs or cell lines referred to in Table 1. Immunospecific binding to PA polypeptides may be determined by immunoassays known in the art or described herein for assaying specific antibody-antigen binding. Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies that specifically bind to PA are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies molecules, fragments and/or variants (SEQ ID NOS:57-65).

In one embodiment of the present invention, antibodies that specifically bind to a PA or a fragment or variant thereof, comprise a polypeptide having the amino acid sequence of a heavy chain of at least one of the scFvs referred to in Table 1 or cell lines contained in the ATCC™ Deposits referred to in Table 1 and/or a light chain of at least one of the scFvs referred to in Table 1 or cell lines contained in the ATCC™ Deposits referred to in Table 1.

In another embodiment of the present invention, antibodies that specifically bind to PA or a fragment or variant thereof, comprise a polypeptide having the amino acid sequence of any one of the VH domains of at least one of the scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1 and/or any one of the VL domains of at least one of the scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1. In preferred embodiments, antibodies of the present invention comprise the amino acid sequence of a VH domain and VL domain from a single scFv referred to in Table 1 or single recombinant antibody expressed by a cell line contained in an ATCC™ Deposit referred to in Table 1. In alternative embodiments, antibodies of the present invention comprise the amino acid sequence of a VH domain and a VL domain from different scFvs referred to in Table 1 or different recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1. Molecules comprising, or alternatively consisting of, antibody fragments or variants of the VH and/or VL domains of at least one of the scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1 that specifically bind to PA are also encompassed by the invention, as are nucleic acid molecules encoding these VH and VL domains, molecules, fragments and/or variants (SEQ ID NOS:57-65).

The present invention also provides antibodies that specifically bind to a polypeptide, or polypeptide fragment or variant of PA, wherein said antibodies comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one, two, three, or more of the VH CDRs contained in a VH domain of one or more scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1. In particular, the invention provides antibodies that specifically bind PA or fragments or variants thereof, comprising, or alternatively consisting of, a polypeptide having the amino acid sequence of a VH CDR1 contained in a VH domain of one or more scFvs or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1. In another embodiment, antibodies that specifically bind PA, comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VH CDR2 contained in a VH domain of one or more scFvs referred to in Table 1 or one or more recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1. In a preferred embodiment, antibodies that specifically bind PA or fragments or variants thereof, comprise, or alternatively consist of a polypeptide having the amino acid sequence of a VH CDR3 contained in a VH domain of one or more scFvs referred to in Table 1 or one or more recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1. Molecules comprising, or alternatively consisting of, these antibodies, or antibody fragments or variants thereof, that specifically bind to PA or a PA fragment or variant thereof are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments and/or variants (SEQ ID NOS:57-65).

The present invention also provides antibodies that specifically bind to a PA polypeptide or a polypeptide fragment or variant of PA, wherein said antibodies comprise, or alternatively consist of, a polypeptide having an amino acid sequence of any one, two, three, or more of the VL CDRs contained in a VL domain of one or more scFvs referred to in Table 1 or one or more recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1. In particular, the invention provides antibodies that specifically bind PA or a fragment or variant thereof, comprising, or alternatively consisting of, a polypeptide having the amino acid sequence of a VL CDR1 contained in a VL domain of one or more scFvs referred to in Table 1 or one or more recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1. In another embodiment, antibodies that specifically bind PA or a fragment or variant thereof, comprise, or alternatively consist of, a polypeptide having the amino acid sequence of a VL CDR2 contained in a VL domain of one or more scFvs referred to in Table 1 or one or more recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1. In a preferred embodiment, antibodies that specifically bind PA or a fragment or variant thereof, comprise, or alternatively consist of a polypeptide having the amino acid sequence of a VL CDR3 contained in a VL domain of one or more scFvs referred to in Table 1 or one or more recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1. Molecules comprising, or alternatively consisting of, these antibodies, or antibody fragments or variants thereof, that specifically bind to PA or a PA fragment or variant thereof are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments and/or variants (SEQ ID NOS:57-65).

The present invention also provides antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants) that specifically bind to PA polypeptide or a fragment or variant of a PA, wherein said antibodies comprise, or alternatively consist of, one, two, three, or more VH CDRs and one, two, three or more VL CDRs, as contained in a VH domain or VL domain of one or more scFvs referred to in Table 1 or one or more recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1. In particular, the invention provides for antibodies that specifically bind to a PA polypeptide or polypeptide fragment or variant of PA, wherein said antibodies comprise, or alternatively consist of, a VH CDR1 and a VL CDR1, a VH CDR1 and a VL CDR2, a VH CDR1 and a VL CDR3, a VH CDR2 and a VL CDR1, VH CDR2 and VL CDR2, a VH CDR2 and a VL CDR3, a VH CDR3 and a VH CDR1, a VH CDR3 and a VL CDR2, a VH CDR3 and a VL CDR3, or any combination thereof, of the VH CDRs and VL CDRs contained in a VH domain or VL domain of one or more scFvs referred to in Table 1 or one or more recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1. In a preferred embodiment, one or more of these combinations are from the same scFv or the same recombinant antibody expressed by cell line contained in an ATCC™ deposit as disclosed in Table 1. Molecules comprising, or alternatively consisting of, fragments or variants of these antibodies, that specifically bind to PA or a fragment or variant thereof are also encompassed by the invention, as are nucleic acid molecules encoding these antibodies, molecules, fragments or variants (SEQ ID NOS:57-65).

In a specific embodiment, a nucleic acid molecule of the invention encodes an antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), comprising, or alternatively consisting of, a VH domain having an amino acid sequence of any one of the VH domains of at least one of the scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1 and a VL domain having an amino acid sequence of VL domain of at least one of the scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1. In another embodiment, a nucleic acid molecule of the invention encodes an antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), comprising, or alternatively consisting of, a VH domain having an amino acid sequence of any one of the VH domains of at least one of the scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1 or a VL domain having an amino acid sequence of a VL domain of at least one of the scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1.

The present invention also provides antibodies that comprise, or alternatively consist of, variants (including derivatives) of the antibody molecules (e.g., the VH domains and/or VL domains) described herein, which antibodies specifically bind to PA or a fragment or variant thereof. Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a molecule of the invention, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which result in amino acid substitutions. Preferably, the variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the reference VH domain, VHCDR1, VHCDR2, VHCDR3, VL domain, VLCDR1, VLCDR2, or VLCDR3. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity (e.g., the ability to bind PA).

For example, it is possible to introduce mutations only in framework regions or only in CDR regions of an antibody molecule. Introduced mutations may be silent or neutral missense mutations, i.e., have no, or little, effect on an antibody's ability to bind antigen. These types of mutations may be useful to optimize codon usage, or improve a hybridoma's antibody production. Alternatively, non-neutral missense mutations may alter an antibody's ability to bind antigen. The location of most silent and neutral missense mutations is likely to be in the framework regions, while the location of most non-neutral missense mutations is likely to be in CDR, though this is not an absolute requirement. One of skill in the art would be able to design and test mutant molecules with desired properties such as no alteration in antigen binding activity or alteration in binding activity (e.g, improvements in antigen binding activity or change in antibody specificity). Following mutagenesis, the encoded protein may routinely be expressed and the functional and/or biological activity of the encoded protein, (e.g., ability to specifically bind PA) can be determined using techniques described herein or by routinely modifying techniques known in the art.

In a specific embodiment, an antibody of the invention (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof), that specifically binds PA or a fragment or variant thereof, comprises, or alternatively consists of, an amino acid sequence encoded by a nucleotide sequence that hybridizes to a nucleotide sequence that is complementary to that encoding one of the VH or VL domains of one or more scFvs referred to in Table 1 or one or more recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1 under stringent conditions, e.g., hybridization to filter-bound DNA in 6× sodium chloride/sodium citrate (SSC) at about 45° C. followed by one or more washes in 0.2×SSC/0.1% SDS at about 50-65° C., under highly stringent conditions, e.g., hybridization to filter-bound nucleic acid in 6×SSC at about 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about 68° C., or under other stringent hybridization conditions which are known to those of skill in the art (see, for example, Ausubel, F. M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3). The nucleic acid molecules encoding these antibodies are also encompassed by the invention.

It is well known within the art that polypeptides, or fragments or variants thereof, with similar amino acid sequences often have similar structure and many of the same biological activities. Thus, in one embodiment, an antibody (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof), that specifically binds to PA or fragments or variants of PA, comprises, or alternatively consists of, a VH domain having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to the amino acid sequence of a VH domain of at least one of the scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1.

In another embodiment, an antibody (including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof), that specifically binds to PA or a fragment or variant of PA, comprises, or alternatively consists of, a VL domain having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to the amino acid sequence of a VL domain of at least one of the scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1.

Methods of Producing Antibodies

Antibodies in accordance with the invention were prepared via the utilization of a phage scFv display library. Technologies utilized for achieving the same are disclosed in the patents, applications, and references disclosed herein.

In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of lymphoid tissues) or synthetic cDNA libraries. The DNA encoding the VH and VL domains are joined together by an scFv linker by PCR and cloned into a phagemid vector (e.g., pCANTAB 6 or pComb 3 HSS). The vector is electroporated in E. coli and the E. coli is infected with helper phage. Phage used in these methods are typically filamentous phage including fd and M13 and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII. Phage expressing an antigen binding domain that binds to an antigen of interest (i.e., a PA polypeptide or a fragment thereof) can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to make the antibodies of the present invention include, but are not limited to, those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT application No. PCT/GB91/O1 134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18719; WO 93/1 1236; WO 95/15982; WO 95/20401; WO97/13844; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,717; 5,780,225; 5,658,727; 5,735,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

For some uses, such as for in vitro affinity maturation of an antibody of the invention, it may be useful to express the VH and VL domains of one or more scFvs referred to in Table 1 or one or more recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1 as single chain antibodies or Fab fragments in a phage display library. For example, the cDNAs encoding the VH and VL domains of the scFvs referred to in Table 1 or recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1 may be expressed in all possible combinations using a phage display library, allowing for the selection of VH/VL combinations that bind PA polypeptides with preferred binding characteristics such as improved affinity or improved off rates. Additionally, VH and VL segments—and in particular, the CDR regions of the VH and VL domains of the scFvs referred to in Table 1 or recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1, in particular, may be mutated in vitro. Expression of V11 and VL domains with “mutant” CDRs in a phage display library allows for the selection of VH/VL combinations that bind PA polypeptides with preferred binding characteristics such as improved affinity or improved off rates.

In particular embodiments, antibodies of the invention comprise the VH and VL domains of the PWD0587 scFv wherein the V11 domain contains one or more of the following mutations (using amino acid numbering according to that of SEQ ID NO:53): Q13R, S31W, I100V, and/or E105D. An antibody comprising the PWD0587 VH domain with the Q13R, S31W and I100V mutations and the PWD0587 VL domain, had an approximately 11 fold increase in affinity for the PA antigen compared to an antibody comprising the PWD0587 heavy and light chains. Thus, in specific embodiments, an antibody of the invention comprises the PWD0587 VH domain with the Q13R, S31W and I100V mutations and the PWD0587 VL domain.

An antibody comprising the PWD0587 VH domain with the Q13R and S31W mutations and the PWD0587 VL domain, had an approximately 68 fold increase in affinity for the PA antigen compared to an antibody comprising the PWD0587 heavy and light chains. Thus, in specific embodiments, an antibody of the invention comprises the PWD0587 VH domain with the Q13R and S31W mutations and the PWD0587 VL domain.

An antibody comprising the PWD0587 VH domain with the Q13R, S31W, I100V and E105D mutations and the PWD0587 VL domain, had an approximately 121 fold increase in affinity for the PA antigen compared to an antibody comprising the PWD0587 heavy and light chains. Thus, in specific embodiments, an antibody of the invention comprises the PWD0587 VH domain with the Q13R, S31W, 1100V and E105D mutations and the PWD0587 VL domain.

An antibody comprising the PWD0587 VH domain with the Q13R, S31W and E105D mutations and the PWD0587 VL domain, had an approximately 665 fold increase in affinity for the PA antigen compared to an antibody comprising the PWD0587 heavy and light chains. Thus in specific embodiment an antibody of the invention comprises the PWD0587 VH domain with the Q13R, S31W and E105D mutations and the PWD0587 VL domain.

Preliminary testing of the four mutant forms of the PWD0587 antibody with increased affinities for PA compared to the parental PWD0587 (unmutated) antibody, indicated that the mutant PWD0587 antibodies behaved comparably to the parental PWD0587 antibody in, for example, a rubidium release assay (e.g., similar to the assays described in Example 5). In a rat lethal toxin challenge model (similar to the assays described in Example 9) an antibody comprising the PWD0587 VH domain with the Q13R, S31 W and E105D mutations and the PWD0587 VL domain was slightly more effective than the parental PWD0587 antibody in preventing lethal toxin induced death.

Additional Methods of Producing Antibodies

Antibodies of the invention (including antibody fragments or variants) can be produced by any method known in the art. For example, it will be appreciated that antibodies in accordance with the present invention can be expressed in cell lines including, but not limited to, myeloma cell lines and hybridoma cell lines. Sequences encoding the cDNAs or genomic clones for the particular antibodies can be used for transformation of a suitable mammalian or nonmammalian host cells or to generate phage display libraries, for example. Additionally, polypeptide antibodies of the invention may be chemically synthesized or produced through the use of recombinant expression systems.

One way to produce the antibodies of the invention would be to clone the VH and/or VL domains of an scFv referred to in Table 1 or recombinant antibody expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1. In order to isolate the VH and VL domains from bacteria transfected with a vector containing the scFv, PCR primers complementary to VH or VL nucleotide sequences (See Example 6), may be used to amplify the VH and VL sequences. The PCR products may then be cloned using vectors, for example, which have a PCR product cloning site consisting of a 5′ and 3′ single T nucleotide overhang, that is complementary to the overhanging single adenine nucleotide added onto the 5′ and 3′ end of PCR products by many DNA polymerases used for PCR reactions. The VH and VL domains can then be sequenced using conventional methods known in the art. Alternatively, the VH and VL domains may be amplified using vector specific primers designed to amplify the entire scFv, (i.e. the VH domain, linker and VL domain.)

The cloned VH and VL genes may be placed into one or more suitable expression vectors. By way of non-limiting example, PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site may be used to amplify the VH or VL sequences. Utilizing cloning techniques known to those of skill in the art, the PCR amplified VH domains may be cloned into vectors expressing the appropriate immunoglobulin constant region, e.g., the human IgG1 or IgG4 constant region for VH domains, and the human kappa or lambda constant regions for kappa and lambda VL domains, respectively. Preferably, the vectors for expressing the VH or VL domains comprise a promoter suitable to direct expression of the heavy and light chains in the chosen expression system, a secretion signal, a cloning site for the immunoglobulin variable domain, immunoglobulin constant domains, and a selection marker such as neomycin. The VH and VL domains may also be cloned into a single vector expressing the necessary constant regions. The heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques known to those of skill in the art (See, for example, Guo et al., J. Clin. Endocrinol. Metab. 82:925-31 (1997), and Ames et al., J. Immunol. Methods 184:177-86 (1995) which are herein incorporated in their entireties by reference).

The invention provides polynucleotides comprising, or alternatively consisting of, a nucleotide sequence encoding an antibody of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof). The invention also encompasses polynucleotides that hybridize under high stringency, or alternatively, under intermediate or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides complementary to nucleic acids having a polynucleotide sequence that encodes an antibody of the invention or a fragment or variant thereof.

The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. If the amino acid sequences of the VH domains, VL domains and CDRs thereof, are known, nucleotide sequences encoding these antibodies can be determined using methods well known in the art, i.e., the nucleotide codons known to encode the particular amino acids are assembled in such a way to generate a nucleic acid that encodes the antibody, of the invention. Such a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells or Epstein Barr virus transformed B cell lines that express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence of the antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.

In a specific embodiment, VH and VL domains of one or more scFvs referred to in Table 1 or one or more recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1, or fragments or variants thereof, are inserted within framework regions using recombinant DNA techniques known in the art. In a specific embodiment, one, two, three, four, five, six, or more of the CDRs of a VH and/or a VL domain of one or more scFvs referred to in Table 1 or one or more recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1, or fragments or variants thereof, are inserted within framework regions using recombinant DNA techniques known in the art. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of human framework regions, the contents of which are hereby incorporated by reference in its entirety). Preferably, the polynucleotides generated by the combination of the framework regions and CDRs encode an antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that specifically binds to a PA polypeptide. Preferably, as discussed supra, polynucleotides encoding variants of antibodies or antibody fragments having one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions do not significantly alter binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules, or antibody fragments or variants, lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and fall within the ordinary skill of the art.

The ability to clone and reconstruct megabase-sized human loci in YACs and to introduce them into the mouse germline provides a powerful approach to elucidating the functional components of very large or crudely mapped loci as well as generating useful models of human disease. Furthermore, the utilization of such technology for substitution of mouse loci with their human equivalents could provide unique insights into the expression and regulation of human gene products during development, their communication with other systems, and their involvement in disease induction and progression.

An important practical application of such a strategy is the “humanization” of the mouse humoral immune system. Introduction of human immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been inactivated offers the opportunity to study the mechanisms underlying programmed expression and assembly of antibodies as well as their role in B cell development. Furthermore, such a strategy could provide an ideal source for production of fully human monoclonal antibodies (mAbs) an important milestone towards fulfilling the promise of antibody therapy in human disease.

Fully human antibodies are expected to minimize the immunogenic and allergic responses intrinsic to mouse or mouse-derivatized Monoclonal antibodies and thus to increase the efficacy and safety of the administered antibodies. The use of fully human antibodies can be expected to provide a substantial advantage in the treatment of chronic and recurring human diseases, such as cancer, which require repeated antibody administrations.

One approach towards this goal was to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci in anticipation that such mice would produce a large repertoire of human antibodies in the absence of mouse antibodies. Large human Ig fragments would preserve the large variable gene diversity as well as the proper regulation of antibody production and expression. By exploiting the mouse machinery for antibody diversification and selection and the lack of immunological tolerance to human proteins, the reproduced human antibody repertoire in these mouse strains should yield high affinity antibodies against any antigen of interest, including human antigens. Using the hybridoma technology, antigen-specific human Monoclonal antibodies with the desired specificity could be readily produced and selected.

This general strategy was demonstrated in connection with the generation of the first XENOMOUSE™ transgenic mouse system strains as published in 1994. See Green et al. Nature Genetics 7:13-21 (1994). The XENOMOUSE™ transgenic mouse system strains were engineered with yeast artificial chromosomes (YACS) containing germline configuration fragments of the human heavy chain locus and kappa light chain locus, respectively, which contained core variable and constant region sequences. Id. The human Ig containing YACs proved to be compatible with the mouse system for both rearrangement and expression of antibodies and were capable of substituting for the inactivated mouse Ig genes. This was demonstrated by their ability to induce B-cell development, to produce an adult-like human repertoire of fully human antibodies, and to generate antigen-specific human monoclonal antibodies. These results also suggested that introduction of larger portions of the human Ig loci containing greater numbers of V genes, additional regulatory elements, and human Ig constant regions might recapitulate substantially the full repertoire that is characteristic of the human humoral response to infection and immunization. The work of Green et al. was recently extended to the introduction of greater than approximately 80% of the human antibody repertoire through introduction of megabase sized, germline configuration YAC fragments of the human heavy chain loci and kappa light chain loci, respectively, to produce XENOMOUSE™ transgenic mouse system mice. See Mendez et al. Nature Genetics 15:146-156 (1997), Green and Jakobovits J. Exp. Med. 188:483-495 (1998), Green, Journal of Immunological Methods 231:11-23 (1999) and U.S. patent application Ser. No. 08/759,620, filed Dec. 3, 1996, the disclosures of which are hereby incorporated by reference.

Human anti-mouse antibody (HAMA) responses have led the industry to prepare chimeric or otherwise humanized antibodies. While chimeric antibodies have a human constant region and a murine variable region, it is expected that certain human anti-chimeric antibody (HACA) responses will be observed, particularly in chronic or multi-dose utilizations of the antibody. Thus, it would be desirable to provide fully human antibodies against PA polypeptides in order to vitiate concerns and/or effects of HAMA or HACA responses.

Monoclonal antibodies specific for PA polypeptides may be prepared using hybridoma technology. (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 571-681 (1981)). Briefly, XENOMOUSE™ transgenic mouse system mice may be immunized with PA polypeptides. After immunization, the splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line, such as the myeloma cell line (SP2O), available from the ATCC™, may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP2O), available from the ATCC™. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981)). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the PA polypeptides.

For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use human or chimeric antibodies. Completely human antibodies are particularly desirable for therapeutic treatment of human patients. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50435, WO 98/24893, WO98/16654, WO 96/34096, WO 96/35735, and WO 91/10741; each of which is incorporated herein by reference in its entirety. In a specific embodiment, antibodies of the present invention comprise one or more VH and VL domains of the invention and constant regions from another immunoglobulin molecule, preferably a human immunoglobulin molecule. In a specific embodiment, antibodies of the present invention comprise one or more CDRs corresponding to the VH and VL domains of the invention and framework regions from another immunoglobulin molecule, preferably a human immunoglobulin molecule. In other embodiments, an antibody of the present invention comprises one, two, three, four, five, six or more VL CDRs or VH CDRs corresponding to one or more of the VH or VL domains of one or more scFvs referred to in Table 1 or one or more recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1, or fragments or variants thereof, and framework regions (and, optionally one or more CDRs not present in the scFvs referred to in Table 1 or recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1) from a human immunoglobulin molecule. In a preferred embodiment, an antibody of the present invention comprises a VH CDR3, VL CDR3, or both, corresponding to the same recombinant antibody, or different recombinant antibodies selected from the recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1, or fragments or variants thereof, and framework regions from a human immunoglobulin.

A chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such as antibodies having a human variable region and a non-human (e.g., murine) immunoglobulin constant region or vice versa. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods 125:191-202 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entirety. Chimeric antibodies comprising one or more CDRs from human species and framework regions from a non-human immunoglobulin molecule (e.g., framework regions from a murine, canine or feline immunoglobulin molecule) (or vice versa) can be produced using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,352). In a preferred embodiment, chimeric antibodies comprise a human CDR3 having an amino acid sequence of any one of the VH CDR3s or VL CDR3s of a VH or VL domain of one or more of the scFvs referred to in Table 1, or a variant thereof, and non-human framework regions or human framework regions different from those of the frameworks in the corresponding scFv disclosed in Table 1. Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 352:323 (1988), which are incorporated herein by reference in their entireties.)

Intrabodies are antibodies, often scFvs, that are expressed from a recombinant nucleic acid molecule and engineered to be retained intracellularly (e.g., retained in the cytoplasm, endoplasmic reticulum, or periplasm). Intrabodies may be used, for example, to ablate the function of a protein to which the intrabody binds. The expression of intrabodies may also be regulated through the use of inducible promoters in the nucleic acid expression vector comprising the intrabody. Intrabodies of the invention can be produced using methods known in the art, such as those disclosed and reviewed in Chen et al., Hum. Gene Ther. 5:595-601 (1994); Marasco, W. A., Gene Ther. 4:11-15 (1997); Rondon and Marasco, Annu. Rev. Microbiol. 51:257-283 (1997); Proba et al., J. Mol. Biol. 275:245-253 (1998); Cohen et al., Oncogene 17:2445-2456 (1998); Ohage and Steipe, J. Mol. Biol. 291:1119-1128 (1999); Ohage et al., J. Mol. Biol. 291:1129-1134 (1999); Wirtz and Steipe, Protein Sci. 8:2245-2250 (1999); Zhu et al., J. Immunol. Methods 231:207-222 (1999); and references cited therein.

Recombinant expression of an antibody of the invention (including antibody fragments or variants thereof (e.g., a heavy or light chain of an antibody of the invention), requires construction of an expression vector(s) containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule (e.g., a whole antibody, a heavy or light chain of an antibody, or portion thereof (preferably, but not necessarily, containing the heavy or light chain variable domain)), of the invention has been obtained, the vector(s) for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention (e.g., a whole antibody, a heavy or light chain of an antibody, a heavy or light chain variable domain of an antibody, or a portion thereof, or a heavy or light chain CDR, a single chain Fv, or fragments or variants thereof), operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464, the contents of each of which are hereby incorporated by reference in its entirety) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy chain, the entire light chain, or both the entire heavy and light chains.

The expression vector(s) is(are) transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing polynucleotide(s) encoding an antibody of the invention (e.g., whole antibody, a heavy or light chain thereof, or portion thereof, or a single chain antibody, or a fragment or variant thereof), operably linked to a heterologous promoter. In preferred embodiments, for the expression of entire antibody molecules, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO 1. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione 5-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) may be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. Antibody coding sequences may be cloned individually into non-essential regions (for example, the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example, the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 8 1:355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., Methods in Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, HeLa, COS, NSO, MDCK, 293, 3T3, W138, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT2O and T47D, and normal mammary gland cell line such as, for example, CRL7O3O and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compositions that interact directly or indirectly with the antibody molecule.

The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, “The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells” in DNA Cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the coding sequence of the antibody, production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

Vectors which use glutamine synthase (GS) or DHFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively. An advantage of glutamine synthase based vectors are the availability of cell lines (e.g., the murine myeloma cell line, NSO) which are glutamine synthase negative. Glutamine synthase expression systems can also function in glutamine synthase expressing cells (e.g. Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene. A glutamine synthase expression system and components thereof are detailed in PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; and WO91/06657 which are incorporated in their entireties by reference herein. Additionally, glutamine synthase expression vectors that may be used according to the present invention are commercially available from suppliers, including, for example Lonza Biologics, Inc. (Portsmouth, N.H.). Expression and production of monoclonal antibodies using a GS expression system in murine myeloma cells is described in Bebbington et al., Bio/technology 10:169 (1992) and in Biblia and Robinson Biotechnol. Prog. 11:1 (1995) which are incorporated in their entireties by reference herein.

The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain is preferably placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2 197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) has been chemically synthesized or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, or more generally, a protein molecule, such as, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the antibodies of the present invention may be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.

Antibodies of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the antibodies of the present invention may be glycosylated or may be non-glycosylated. In addition, antibodies of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.

The invention encompasses antibodies which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.

Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression. The antibodies may also be modified with a detectable label, such as an enzymatic, fluorescent, radioisotopic or affinity label to allow for detection and isolation of the antibody.

In specific embodiments, antibodies of the invention may be labeled with Europium. For example, antibodies of the invention may be labelled with Europium using the DELFIA Eu-labeling kit (catalog #1244-302, Perkin Elmer Life Sciences, Boston, Mass.) following manufacturer's instructions.

In specific embodiments, antibodies of the invention are attached to macrocyclic chelators useful for conjugating radiometal ions, including but not limited to, 111In, 177Lu, 90Y, 166Ho, 153Sm, 215Bi and 225Ac to polypeptides. In a preferred embodiment, the radiometal ion associated with the macrocyclic chelators attached to antibodies of the invention is 111In. In another preferred embodiment, the radiometal ion associated with the macrocyclic chelator attached to antibodies polypeptides of the invention is 90Y. In specific embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA). In specific embodiments, the macrocyclic chelator is α-(5-isothiocyanato-2-methoxyphenyl)-1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid. In other specific embodiments, the DOTA is attached to the antibody of the invention via a linker molecule. Examples of linker molecules useful for conjugating a macrocyclic chelator such as DOTA to a polypeptide are commonly known in the art—see, for example, DeNardo et al., Clin Cancer Res. 4(10):2483-90, 1998; Peterson et al., Bioconjug. Chem. 10(4):553-7, 1999; and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50, 1999 which are hereby incorporated by reference in their entirety. In addition, U.S. Pat. Nos. 5,652,361 and 5,756,065, which disclose chelating agents that may be conjugated to antibodies, and methods for making and using them, are hereby incorporated by reference in their entireties.

In one embodiment, antibodies of the invention are labeled with biotin. In other related embodiments, biotinylated antibodies of the invention may be used, for example, as an imaging agent or as a means of identifying one or more TRAIL receptor coreceptor or ligand molecules.

Also provided by the invention are chemically modified derivatives of antibodies of the invention which may provide additional advantages such as increased solubility, stability and in vivo or in vitro circulating time of the polypeptide, or decreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The antibodies may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog). For example, the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

The polyethylene glycol molecules (or other chemical moieties) should be attached to the antibody with consideration of effects on functional or antigenic domains of the antibody. There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include, for example, lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues, glutamic acid residues, and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins, e.g., antibodies, via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to a proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.

One may specifically desire antibodies chemically modified at the N-terminus of either the heavy chain or the light chain or both. Using polyethylene glycol as an illustration, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective chemical modification at the N-terminus may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.

As indicated above, pegylation of the antibodies of the invention may be accomplished by any number of means. For example, polyethylene glycol may be attached to the antibody either directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998); U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by reference.

One system for attaching polyethylene glycol directly to amino acid residues of antibodies without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (CISO2CH2CF3). Upon reaction of protein with tresylated MPEG, polyethylene glycol is directly attached to amine groups of the protein. Thus, the invention includes antibody-polyethylene glycol conjugates produced by reacting antibodies of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.

Polyethylene glycol can also be attached to antibodies using a number of different intervening linkers. For example, U.S. Pat. No. 5,612,460, the entire disclosure of which is incorporated herein by reference, discloses urethane linkers for connecting polyethylene glycol to proteins. Antibody-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the antibody by a linker can also be produced by reaction of antibodies with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. A number additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in WO 98/32466, the entire disclosure of which is incorporated herein by reference. Pegylated antibody products produced using the reaction chemistries set out herein are included within the scope of the invention.

The number of polyethylene glycol moieties attached to each antibody of the invention (i.e., the degree of substitution) may also vary. For example, the pegylated antibodies of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per antibody molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

Characterization of Anti-PA Antibodies

Antibodies of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) may also be described or specified in terms of their binding to PA polypeptides or fragments or variants of PA polypeptides. In specific embodiments, antibodies of the invention bind PA polypeptides, or fragments or variants thereof, with a dissociation constant or KD of less than or equal to 5×10−2 M, 10−2 M, 5×10−3 M, 10−3 M, 5×10−4 M, 10−4 M, 5×10−5 M, or 10−5 M. More preferably, antibodies of the invention bind PA polypeptides or fragments or variants thereof with a dissociation constant or KD less than or equal to 5×10−6 M, 10−6 M, 5×10−7 M, 10−7M, 5×10−8 M, or 10−8 M. Even more preferably, antibodies of the invention bind PA polypeptides or fragments or variants thereof with a dissociation constant or KD less than or equal to 5×10−9 M, 5×10−9 M, 10−10 M, 5×10−11 M, 10−11 M, 5×10−12 M, 10−12 M, 5×−13 M, 10−13 M, 5×10−14 M, 10−14 M, 5×10−15 M, or 10−15 M. The invention encompasses antibodies that bind PA polypeptides with a dissociation constant or KD that is within any one of the ranges that are between each of the individual recited values.

In specific embodiments, antibodies of the invention bind PA polypeptides or fragments or variants thereof with an off rate (koff) of less than or equal to 5×10−2 sec−1, 10−2 sec−1, 5×10−3 sec−1 or 10−3 sec−1. More preferably, antibodies of the invention bind PA polypeptides or fragments or variants thereof with an off rate (koff) less than or equal to 5×10−4 sec−1, 10−4 sec−1, 5×10−5 sec−1, or 10−5 sec−1, 5×10−6 sec−1, 10−6 sec−1, 5×10−7 sec−1 or 10−7 sec−1. The invention encompasses antibodies that bind PA polypeptides with an off rate (koff) that is within any one of the ranges that are between each of the individual recited values.

In other embodiments, antibodies of the invention bind PA polypeptides or fragments or variants thereof with an on rate (kon) of greater than or equal to 103 M−1 sec−1, 5×103 M−1 sec−1, 104 M−1 sec−1 or 5×104 M−1 sec−1. More preferably, antibodies of the invention bind PA polypeptides or fragments or variants thereof with an on rate (kon) greater than or equal to 105 M−1 sec−1, 5×105 M−1 sec−1, 106 M−1 sec−1, or 5×106 M−1 sec−1 or 107M−1 sec−1. The invention encompasses antibodies that bind PA polypeptides with on rate (kon) that is within any one of the ranges that are between each of the individual recited values.

In preferred embodiments, the antibodies of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), specifically bind to PA polypeptides and do not cross-react with any other antigens. In preferred embodiments, the antibodies of the invention specifically bind to PA polypeptides (e.g., SEQ ID NO:2 or fragments or variants thereof) and do not cross-react with other bacterial binary toxins (A-B toxins) such as those from Clostridum difficile, Clostridium perfringens, Clostridium spiroforme, Clostridium botulinum, Bacillus cereus and/or Bacillus thuringiensis.

In another embodiment, the antibodies of the present invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), specifically bind to PA polypeptides and cross-react with other antigens. In other embodiments, the antibodies of the invention specifically bind to PA polypeptides (e.g., SEQ ID NO:2 or fragments or variants thereof) and cross-react with other bacterial binary toxins (A-B toxins) such as those from Clostridum difficile, Clostridium perfringens, Clostridium spiroforme, Clostridium botulinum, Bacillus cereus and/or Bacillus thuringiensis.

In a preferred embodiment, antibodies of the invention preferentially bind PA (SEQ ID NO:2), or fragments and variants thereof relative to their ability to bind other antigens (e.g., other bacterial binary toxins (A-B toxins) such as those from Clostridum difficile, Clostridium perfringens, Clostridium spiroforme, Clostridium botulinum, Bacillus cereus and/or Bacillus thuringiensis). An antibody's ability to preferentially bind one antigen compared to another antigen may be determined using any method known in the art.

By way of non-limiting example, an antibody may be considered to bind a first antigen preferentially if it binds said first antigen with a dissociation constant (KD) that is less than the antibody's KD for the second antigen.

In another non-limiting embodiment, an antibody may be considered to bind a first antigen preferentially if it binds said first antigen with an affinity (i.e., KD) that is at least one order of magnitude less than the antibody's KD for the second antigen. In another non-limiting embodiment, an antibody may be considered to bind a first antigen preferentially if it binds said first antigen with an affinity (i.e., KD) that is at least two orders of magnitude less than the antibody's KD for the second antigen.

In another non-limiting embodiment, an antibody may be considered to bind a first antigen preferentially if it binds said first antigen with an off rate (koff) that is less than the antibody's koff for the second antigen. In another non-limiting embodiment, an antibody may be considered to bind a first antigen preferentially if it binds said first antigen with a koff that is at least one order of magnitude less than the antibody's koff for the second antigen. In another non-limiting embodiment, an antibody may be considered to bind a first antigen preferentially if it binds said first antigen with a koff that is at least two orders of magnitude less than the antibody's koff for the second antigen.

The invention also encompasses antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) that have one or more of the same biological characteristics as one or more of the antibodies described herein. By “biological characteristics” is meant, the in vitro or in vivo activities or properties of the antibodies, such as, for example, the ability to bind to PA polypeptides (e.g., either the PA83 or PA63 form of PA); or the ability to inhibit the cleavage of the PA83 into PA20 and PA63 by proteases such as trypsin or furin. Additionally, antibodies of the invention may: prevent oligomerization of PA63, especially heptamerization of PA63; inhibit or abolish the ability of PA63 to bind to an anthrax receptor, e.g., ATR and/or CMG2 (See Example 3); inhibit or abolish the ability of PA63 to bind LF or EF; inhibit or abolish the ability of PA63 to form pores in membranes (see Example 5); inhibit or abolish the ability of lethal toxin (LT) to kill cells, such as macrophages (see Example 8), or animals (see Examples 9-12); or inhibit or abolish the ability of PA heptamers to translocate LF or EF across a membrane (see Example 13). Optionally, the antibodies of the invention will bind to the same epitope as at least one of the antibodies specifically referred to herein. Such epitope binding can be routinely determined using assays known in the art.

The present invention also provides for antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that inhibit or abolish biological activities of PA. By “biological activities of PA” is meant, for example, the ability of PA83 to be cleaved by proteases into PA20 and PA63 fragments; the ability of PA to bind to ATR and/or CMG2; the ability of PA or PA63 to oligomerize, especially to heptamerize; the ability of PA63 to bind LF or EF; the ability of PA63 heptamers to form pores in a membrane; and/or the ability of PA heptamers to translocate EF or LF across a membrane. In one embodiment, an antibody that inhibits or abolishes biological activities of PA comprises, or alternatively consists of a VH and/or a VL domain of at least one of the scFvs referred to in Table 1 or recombinant antibodies expressed by the cell lines referred to in Table 1, or a fragment or variant thereof. In a specific embodiment, an antibody that inhibits or abolishes biological activities of PA comprises, or alternatively consists of a VH and a VL domain of any one of the scFvs referred to in Table 1 or recombinant antibodies expressed by the cell lines referred to in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.

The present invention also provides for antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that inhibit the cleavage of the PA83 into PA20 and PA63 by proteases such as trypsin or furin. See, e.g., Example 2 wherein an antibody that binds peptides that span the RKKR (residues 193-196 of SEQ ID NO:2) cleavage site of PA may be predictive of an antibody's ability to inhibit the cleavage of PA by proteases. Alternatively, a PA cleavage assay is described in J. Biol. Chem. (1992), 267:16396-402, which is hereby incorporated by reference in its entirety. In one embodiment, an antibody that inhibits the cleavage of the PA83 into PA20 and PA63 comprises, or alternatively consists of a VH and/or a VL domain of at least one of the scFvs referred to in Table 1 or recombinant antibodies expressed by the cell lines referred to in Table 1, or a fragment or variant thereof. In a specific embodiment, an antibody that inhibits the cleavage of the PA83 into PA20 and PA63 comprises, or alternatively consists of a VH and a VL domain of any one of the scFvs referred to in Table 1 or recombinant antibodies expressed by the cell lines referred to in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.

The present invention also provides for antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that block or inhibit the binding of PA to ATR and/or CMG2 (e.g., see Example 3). In one embodiment, an antibody that blocks or inhibits the binding of PA to ATR and/or CMG2 comprises, or alternatively consists of a VH and/or a VL domain of at least one of the scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1, or a fragment or variant thereof. In a specific embodiment, an antibody that blocks or inhibits the binding of PA to ATR and/or CMG2 comprises, or alternatively consists of a VH and a VL domain of any one of the scFvs referred to in Table 1 or any one of the recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.

The present invention also provides for antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that block or inhibit the ability of PA or PA63 to heptamerize. In one embodiment, an antibody that blocks or inhibits the ability of PA or PA63 to heptamerize comprises, or alternatively consists of a VH and/or a VL domain of at least one of the scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1, or a fragment or variant thereof. In a specific embodiment, an antibody that blocks or inhibits the ability of PA63 to heptamerize comprises, or alternatively consists of a VH and a VL domain of any one of the scFvs referred to in Table 1 or any one of the recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.

The present invention also provides for antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that block or inhibit the ability of PA63 to bind EF or LF. In one embodiment, an antibody that blocks or inhibits the ability of PA63 to bind EF or LF comprises, or alternatively consists of a VH and/or a VL domain of at least one of the scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1, or a fragment or variant thereof. In a specific embodiment, an antibody that blocks or inhibits the ability of PA63 to bind EF or LF comprises, or alternatively consists of a VH and a VL domain of any one of the scFvs referred to in Table 1 or any one of the recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.

The present invention also provides for antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that block or inhibit the ability of PA63 heptamers to form pores in membranes. In one embodiment, an antibody that blocks or inhibits the ability of PA63 heptamers to form pores in membranes comprises, or alternatively consists of a VH and/or a VL domain of at least one of the scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1, or a fragment or variant thereof. In a specific embodiment, an antibody that blocks or inhibits the ability of PA63 heptamers to form pores in membranes comprises, or alternatively consists of a VH and a VL domain of any one of the scFvs referred to in Table 1 or any one of the recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.

The present invention also provides for antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that block or inhibit the ability of PA63 heptamers to translocate EF or LF across membranes. In one embodiment, an antibody that blocks or inhibits the ability of PA63 heptamers to translocate EF or LF across membranes comprises, or alternatively consists of a VH and/or a VL domain of at least one of the scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1, or a fragment or variant thereof. In a specific embodiment, an antibody that blocks or inhibits the ability of PA63 heptamers to translocate EF or LF across membranes comprises, or alternatively consists of a VH and a VL domain of any one of the scFvs referred to in Table 1 or any one of the recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.

The present invention also provides for antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that block or inhibit the ability of anthrax lethal toxin to kill cells or animals. In one embodiment, an antibody that blocks or inhibits the ability of anthrax lethal toxin to kill cells or animals comprises, or alternatively consists of a VH and/or a VL domain of at least one of the scFvs referred to in Table 1 or at least one of the recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1, or a fragment or variant thereof. In a specific embodiment, an antibody that blocks or inhibits the ability of anthrax lethal toxin to kill cells or animals comprises, or alternatively consists of a VH and a VL domain of any one of the scFvs referred to in Table 1 or any one of the recombinant antibodies expressed by the cell lines contained in the ATCC™ Deposits referred to in Table 1, or a fragment or variant thereof. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.

The present invention also provides for fusion proteins comprising, or alternatively consisting of, an antibody (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), that specifically bind to PA fused to a heterologous polypeptide. Preferably, the heterologous polypeptide to which the antibody is fused is useful for function or is useful to target the fusion protein to cells with surface bound PA molecules. In specific embodiments the invention encompasses bispecific antibodies in which one antibody binding site is specific for PA and the second antibody binding site is specific for a heterologous polypeptide. In one embodiment, a fusion protein of the invention comprises, or alternatively consists of, a polypeptide having the amino acid sequence of any one or more of the VH domains of an antibody of the invention or the amino acid sequence of any one or more of the VL domains of an antibody of the invention or fragments or variants thereof, and a heterologous polypeptide sequence. In another embodiment, a fusion protein of the present invention comprises, or alternatively consists of, a polypeptide having the amino acid sequence of any one, two, three, or more of the VH CDRs of an antibody of the invention, or the amino acid sequence of any one, two, three, or more of the VL CDRs of an antibody of the invention, or fragments or variants thereof, and a heterologous polypeptide sequence. In a preferred embodiment, the fusion protein comprises, or alternatively consists of, a polypeptide having the amino acid sequence of, a VH CDR3 of an antibody of the invention, or fragment or variant thereof, and a heterologous polypeptide sequence, which fusion protein specifically binds to PA. In another embodiment, a fusion protein comprises, or alternatively consists of a polypeptide having the amino acid sequence of at least one VH domain of an antibody of the invention and the amino acid sequence of at least one VL domain of an antibody of the invention or fragments or variants thereof, and a heterologous polypeptide sequence. Preferably, the VH and VL domains of the fusion protein correspond to a single antibody (or scFv or Fab fragment) of the invention. In yet another embodiment, a fusion protein of the invention comprises, or alternatively consists of a polypeptide having the amino acid sequence of any one, two, three or more of the VH CDRs of an antibody of the invention and the amino acid sequence of any one, two, three or more of the VL CDRs of an antibody of the invention, or fragments or variants thereof, and a heterologous polypeptide sequence. Preferably, two, three, four, five, six, or more of the VHCDR(s) or VLCDR(s) correspond to single antibody (or scFv or Fab fragment) of the invention. Nucleic acid molecules encoding these fusion proteins are also encompassed by the invention.

Antibodies of the present invention (including antibody fragments or variants thereof) may be characterized in a variety of ways. In particular, antibodies and related molecules of the invention may be assayed for the ability to specifically bind to PA or a fragment or variant of PA, using techniques described herein or routinely modifying techniques known in the art. Assays for the ability of the antibodies of the invention to specifically bind PA or a fragment or variant of PA, may be performed in solution (e.g., Houghten, Bio/Techniques 13:412-421 (1992)), on beads (e.g., Lam, Nature 354:82-84 (1991)), on chips (e.g., Fodor, Nature 364:555-556 (1993)), on bacteria (e.g., U.S. Pat. No. 5,223,409), on spores (e.g., U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids (e.g., Cull et al., Proc. Natl. Acad. Sci. USA 89:1865-1869 (1992)) or on phage (e.g., Scott and Smith, Science 249:386-390 (1990); Devlin, Science 249:404-406 (1990); Cwirla et al., Proc. Natl. Acad. Sci. USA 87:7178-7182 (1990); and Felici, J. Mol. Biol. 222:301-310 (1991)) (each of these references is incorporated herein in its entirety by reference). Antibodies that have been identified to specifically bind to PA or a fragment or variant of PA can then be assayed for their specificity and affinity for PA using or routinely modifying techniques described herein or otherwise known in the art (see, e.g., Examples 1 and 2).

The antibodies of the invention may be assayed for specific binding to PA polypeptides and cross-reactivity with other antigens by any method known in the art. Immunoassays which can be used to analyze specific binding and cross-reactivity include, but are not limited to, competitive and non-competitive assay systems using techniques such as BIAcore analysis, FACS (fluorescence activated cell sorter) analysis, immunofluorescence, immunocytochemistry, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, western blots, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).

ELISAs comprise preparing antigen, coating the well of a 96-well microtiter plate with the antigen, washing away antigen that did not bind the wells, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the wells and incubating for a period of time, washing away unbound antibodies or non-specifically bound antibodies, and detecting the presence of the antibodies specifically bound to the antigen coating the well. In ELISAs, the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Alternatively, the antigen need not be directly coated to the well; instead the ELISA plates may be coated with an anti-Ig Fc antibody, and the antigen in the form or a PA-Fc fusion protein, may be bound to the anti-Ig Fc coated to the plate. This may be desirable so as to maintain the antigen protein (e.g., the PA polypeptides) in a more native conformation than it may have when it is directly coated to a plate. In another alternative, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, the detectable molecule could be the antigen conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase). One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody (including an scFv or other molecule comprising, or alternatively consisting of, antibody fragments or variants thereof) to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., antigen labeled with 3H or 125I), or fragment or variant thereof with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of the present invention for PA and the binding off-rates can be determined from the data by Scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, a PA polypeptide is incubated with an antibody of the present invention conjugated to a labeled compound (e.g., compound labeled with 3H or 125I) in the presence of increasing amounts of an unlabeled second anti-PA antibody. Assays for determining the ability of one antibody to competitively inhibit the binding of another antibody are known in the art (See, for example, Harlow, Ed & David Lane, Antibodies: A Laboratoiy Manual, New York: Cold Spring Harbor Laboratory, 1988. pp. 567-569.) This kind of competitive assay between two antibodies, may also be used to determine if two antibodies bind the same, closely associated (e.g., overlapping) or different epitopes.

In a preferred embodiment, BIAcore kinetic analysis is used to determine the binding on and off rates of antibodies (including antibody fragments or variants thereof) to PA, or fragments of PA.

Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1 to 4 hours) at 40 degrees C., adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 40 degrees C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

Antibody Conjugates

The present invention encompasses antibodies (including antibody fragments or variants thereof), recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to a heterologous polypeptide (or portion thereof, preferably at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids of the polypeptide) to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. For example, antibodies of the invention may be used to target heterologous polypeptides to particular cell types (e.g., cancer cells), either in vitro or in vivo, by fusing or conjugating the heterologous polypeptides to antibodies of the invention that are specific for particular cell surface antigens or which bind antigens that bind particular cell surface receptors. Antibodies of the invention may also be fused to albumin (including but not limited to recombinant human serum albumin (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporated by reference in their entirety)), resulting in chimeric polypeptides, In a preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with the mature form of human serum albumin (i.e., amino acids 1-585 of human serum albumin as shown in FIGS. 1 and 2 of EP Patent 0 322 094) which is herein incorporated by reference in its entirety. In another preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with polypeptide fragments comprising, or alternatively consisting of, amino acid residues 1-z of human serum albumin, where z is an integer from 369 to 419, as described in U.S. Pat. No. 5,766,883 herein incorporated by reference in its entirety. Polypeptides and/or antibodies of the present invention (including fragments or variants thereof) may be fused to either the N- or C-terminal end of the heterologous protein (e.g., immunoglobulin Fc polypeptide or human serum albumin polypeptide). Polynucleotides encoding fusion proteins of the invention are also encompassed by the invention. Such fusion proteins may, for example, facilitate purification and may increase half-life in vivo. Antibodies fused or conjugated to heterologous polypeptides may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO 93/2 1232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452 (1991), which are incorporated by reference in their entireties.

Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to modulate the activities of antibodies (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof), such methods can be used to generate antibodies with altered activity (e.g., antibodies with higher affinities and lower dissociation rates). See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-35 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents and publications are hereby incorporated by reference in its entirety). In one embodiment, polynucleotides encoding antibodies of the invention may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more portions of a polynucleotide encoding an antibody which portions specifically bind to PA may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

Moreover, the antibodies of the present invention (including antibody fragments or variants thereof), can be fused to marker sequences, such as a polypeptides to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine polypeptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the FLAG® tag (Stratagene, La Jolla, Calif.).

The present invention further encompasses antibodies (including antibody fragments or variants thereof), conjugated to a diagnostic or therapeutic agent. The antibodies can be used, for example, as part of a clinical testing procedure to, e.g., determine the safety or efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include, but are not limited to, streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes, but is not limited to, luminol; examples of bioluminescent materials include, but are not limited to, luciferase, luciferin, and aequorin; and examples of suitable radioactive material include, but are not limited to, iodine (121I, 123I, 125I, 131I), carbon (14C), sulfur (35S), tritium (3H), indium (111In, 112In, 113mIn, 115mIn), technetium (99Tc, 99mTc), thallium (201Ti, gallium (68Ga, 67Ga), palladium (103Pd), molybdenum (99Mo), xenon (135Xe), fluorine (18F), 153Sm, 177Lu, 159Gd, 149Pm, 140La, 175YB, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142Pr, 105Rh, and 97Ru.

Further, an antibody of the invention (including an scFv or other molecule comprising, or alternatively consisting of, antibody fragments or variants thereof), may be coupled or conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi, or other radioisotopes such as, for example, 103Pd, 135Xe, 131I, 68Ge, 57Co, 65Zn, 85Sr, 32P, 35S, 90Y, 153Sm, 153Gd, 169Yb, 51Cr, 54Mn, 75Se, 113Sn, 90Y, 117Tin, 186Re, 188Re and 166Ho. In specific embodiments, an antibody or fragment thereof is attached to macrocyclic chelators that chelate radiometal ions, including but not limited to, 177Lu, 90Y, 166Ho, and 153Sm, to polypeptides. In specific embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA). In other specific embodiments, the DOTA is attached to the an antibody of the invention or fragment thereof via a linker molecule. Examples of linker molecules useful for conjugating DOTA to a polypeptide are commonly known in the art—see, for example, DeNardo et al., Clin Cancer Res. 4(10):2483-90, 1998; Peterson et al., Bioconjug. Chem. 10(4):553-7, 1999; and Zimmerman et al., Nucl. Med. Biol. 26(8):943-50, 1999 which are hereby incorporated by reference in their entirety.

Techniques known in the art may be applied to label antibodies of the invention. Such techniques include, but are not limited to, the use of bifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065; 5,714,711; 5,696,239; 5,652,371; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contents of each of which are hereby incorporated by reference in its entirety) and direct coupling reactions (e.g., Bolton-Hunter and Chloramine-T reaction).

Antibodies of the invention (including antibody fragments or variants thereof), may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Alternatively, an antibody of the invention can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.

An antibody of the invention (including an other molecules comprising, or alternatively consisting of, an antibody fragment or variant thereof), with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.

Uses of Antibodies of the Invention

Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of PA polypeptides in biological and non-biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety). By way of another non-limiting example, antibodies of the invention may be administered to individuals as a form of passive immunization.

Prophylactic or therapeutic treatment with anti-PA antibodies has advantages over other anti-anthrax agents, such as antibiotics, in that anti-PA antibodies provide protection against drug resistant strains; anti-PA antibodies can be given as either a single dose treatment or can be given in multiple doses (e.g., bi-weekly or monthly dosing); individual doses of anti-PA antibodies will have a relatively long duration of effect; can be administered subcutaneously in addition to other routes of administration (e.g., intravenously), and will be useful in re-exposure or flare situations. Given that the anti-PA antibodies provided herein are fully human antibodies, the risk of side effects due to anti-PA treatment will be minimal when administered as fully human antibodies.

Epitope Mapping

The present invention provides antibodies (including antibody fragments or variants thereof), that can be used to identify epitopes of a PA polypeptide (e.g., SEQ ID NO:2)) using techniques described herein or otherwise known in the art. Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985), further described in U.S. Pat. No. 4,711,211.) Identified epitopes of antibodies of the present invention may, for example, be used as vaccine candidates, i.e., to immunize an individual to elicit antibodies against the naturally occurring forms of PA polypeptides.

Diagnostic Uses of Antibodies

Labeled antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) which specifically bind to a PA polypeptide can be used for diagnostic purposes to detect, diagnose, prognose, or monitor the presence of the intact Bacillus anthracis spore or organism, or simply the components of anthrax toxin. In specific embodiments, labeled antibodies of the invention (including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof) which specifically bind to a PA polypeptide can be used for diagnostic purposes to detect, diagnose, prognose, or monitor the course of anthrax infection.

The invention provides for the detection of expression of a PA polypeptide comprising: (a) assaying the expression of a PA polypeptide in a (biological—or non-biological) sample from an individual using one or more antibodies of the invention that specifically binds to PA; and (b) detecting the presence of PA polypeptide in the sample.

The invention provides for the detection of aberrant expression of a PA polypeptide comprising: (a) assaying the expr